Screening Methods

Abstract

Provided is a screening method including: a primary screening step in which plant cells are brought into contact with candidate compounds and then cultured under oxidative stress-inducing conditions and non-oxidative stress-inducing conditions, the generated amount of a reactive oxygen species is measured to perform a primary screening, and primary candidate compounds are selected from the candidate compounds; and a secondary screening step in which plant cells are brought into contact with the primary candidate compounds and then cultured under oxidative stress-inducing conditions and non-oxidative stress-inducing conditions, the viability of the plant cells is measured to perform a secondary screening, and, as a result, secondary candidate compounds are selected from the primary candidate compounds. Also provided are a biostimulant, a prophylactic agent, and a herbicide that contain the compounds selected by using the screening method according to the present invention. The screening method according to the present invention makes it possible to reduce cost, time, and labor as compared with conventional screening methods.

Description

Screening method, biostimulant, disease prevention agent and herbicide Cross Reference

[0001] This application claims priority based on Japanese Patent Application No. 2023-201701 filed on November 29, 2023, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to a screening method for evaluating whether a plurality of candidate compounds can be used as a plant biostimulant, a plant disease prevention agent, or a plant herbicide, and also to a biostimulant, a plant disease prevention agent, or a plant herbicide containing a compound selected by the screening method.

[0003] Conventionally, there has been a wide search for compounds that can be used as active ingredients of agents that affect plants (biostimulants, disease prevention agents, or herbicides). However, in recent years, the number of compounds required to develop one product (i.e., the number of compounds to be screened) has increased significantly, and as a result, the frequency of drug development has decreased significantly.

[0004] For example, in a conventional screening method for disease prevention agents, a candidate compound for the disease prevention agent is administered to actual plants such as rice along with a pathogen, and visual selection is carried out using the spread of lesions as an indicator (see, for example, Patent Document 1). The situation is similar for biostimulants. Furthermore, visual selection, such as checking the state of withering after spraying a compound, is also common in screening methods for herbicides.

[0005] JP 2009-72070 A

[0006] However, conventional screening methods for biostimulants, disease prevention agents or herbicides have the problem of requiring a great deal of cost, time and effort.

[0007] The present invention has been made to solve the above problems, and aims to provide a screening method that can reduce costs, time, and labor compared to conventional screening methods. Another aim of the present invention is to provide biostimulants, disease prevention agents, and herbicides containing compounds selected by the screening method of the present invention.

[0008] [1] The screening method of the present invention is a screening method for evaluating whether or not a plurality of candidate compounds can be used as biostimulants, disease prevention agents, or herbicides for plants, and is characterized by comprising a primary screening step of contacting plant cells with the candidate compounds, then culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the amount of reactive oxygen species produced to perform primary screening, and selecting primary candidate compounds from the candidate compounds; and a secondary screening step of contacting the plant cells with the primary candidate compounds, then culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the viability of the plant cells to perform secondary screening, and selecting secondary candidate compounds from the primary candidate compounds.

[0009] [2] In the screening method described in [1] above, it is preferable that the plant cells are plant cells having photosynthetic ability, and that in the secondary screening step, the viability of the plant cells is measured by measuring autofluorescence derived from chlorophyll.

[0010] [3] In the screening method described in [2] above, the plant cells are preferably T-87 strain green cultured cells derived from Arabidopsis thaliana.

[0011] [4] In the screening method described in [1] above, the substance used under the oxidative stress-inducing conditions is preferably paraquat (methyl viologen).

[0012] [5] In the screening method according to [1] above, it is preferable that the amount of reactive oxygen species produced is measured using a chemiluminescent probe in the primary screening step.

[0013] [6] In the screening method described in [5] above, the chemiluminescent probe is preferably MCLA (methyl cypridina luciferin analog).

[0014] [7] The biostimulant of the present invention is characterized by containing the following compound (1):

[0015] [8] The epidemic prevention agent of the present invention is characterized by containing the following compound (2):

[0016] [9] The herbicide of the present invention is characterized by containing the following compound (3):

[0017] The screening method of the present invention comprises a primary screening step of contacting plant cells with candidate compounds, followed by culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the amount of reactive oxygen species produced, and selecting primary candidate compounds from the candidate compounds, and a secondary screening step of contacting plant cells with the primary candidate compounds, followed by culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the viability of the plant cells, and selecting secondary candidate compounds from the primary candidate compounds. Therefore, the screening method of the present invention can be performed using small amounts of plant cells and compounds, and enables the simple and rapid screening of a wide variety of compounds, thereby reducing costs, time, and labor compared to conventional screening methods.

[0018] Furthermore, the biostimulants, disease prevention agents and herbicides of the present invention are useful drug candidates, including compounds for which new uses have been found by the screening methods of the present invention.

[0019] 1 is a graph showing the dose dependency of reactive oxygen species production and cell death when the T-87 strain was cultured with the addition of paraquat in a preliminary study in the Examples. FIG. 2 is a diagram showing the appearance of a 96-well plate used in the primary screening step and the secondary screening step in the Examples. FIG. 3 is a table showing an example of the results of the primary screening step in the Examples. FIG. 4 is a diagram showing an example of the results of the secondary screening step in the Examples. FIG. 5 is a diagram showing the effect of compound (1) as a biostimulant. FIG. 6 is a bar graph showing the effect of compound (2) as an epidemic prevention agent. FIG. 7 is a diagram showing the effect of compound (3) as a herbicide.

[0020] The screening method, biostimulant, disease prevention agent, and herbicide of the present invention will be described below based on embodiments.

[0021] [Embodiments] 1. Screening Method According to Embodiments The screening method according to embodiments is a screening method for evaluating whether a plurality of candidate compounds can be used as a plant biostimulant, an epidemic prevention agent, or a herbicide. The screening method according to embodiments includes a primary screening step and a secondary screening step.

[0022] As used herein, the term "biostimulant" refers to an agent that stimulates plants to activate their cells and has the effect of protecting them from various abiotic stresses (for example, stresses resulting from temperature, ultraviolet light, physical stimuli, etc.) (eliminating or suppressing the effects of various abiotic stresses).

[0023] In addition, the term "epidemic prevention agent" as used herein refers to an agent that has the effect of protecting a plant from at least one pathogen (preventing or suppressing the effects of a pathogen).

[0024] In addition, the term "herbicide" as used herein refers to a chemical that has the effect of killing or stopping the growth of at least a part of a plant.

[0025] The primary screening step is a step of contacting plant cells with candidate compounds, culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the amount of reactive oxygen species produced, and selecting primary candidate compounds from the candidate compounds. The primary screening step is a step carried out to eliminate compounds that do not affect the increase or decrease of reactive oxygen species under oxidative stress-inducing conditions.

[0026] As used herein, "oxidative stress-inducing conditions" refers to conditions that make it easy for reactive oxygen species to be generated in plant cells. Reactive oxygen species (ROS) is a general term for highly reactive substances that are generated when oxygen captures electrons. Examples of reactive oxygen species include superoxide (O ２ －・ ), hydrogen peroxide (H ２ O ２ ), hydroxyl radical (OH ・ ) and singlet oxygen ( １ O ２ At low concentrations, reactive oxygen species (ROS) not only have direct antibacterial effects but also play multiple beneficial roles within cells as signaling molecules that regulate the expression of stress-responsive resistance genes and various metabolic pathways. On the other hand, at high concentrations, ROS and related redox-active compounds cause cell damage and necrosis through oxidative stress (e.g., oxidation of various biomolecules such as DNA, proteins, and lipids).

[0027] The primary screening is performed from the following two perspectives. The first perspective is whether the amount of reactive oxygen species produced after culture under oxidative stress-non-inducing conditions is significantly less than the amount of reactive oxygen species produced in a control in which plant cells are cultured under oxidative stress-non-inducing conditions without contacting them with a candidate compound. The second perspective is whether the amount of reactive oxygen species produced after culture under oxidative stress-inducing conditions is significantly less or more than the amount of reactive oxygen species produced in a control in which plant cells are cultured under oxidative stress-inducing conditions without contacting them with a candidate compound. The primary candidate compound can be said to be an reactive oxygen regulator that affects the increase or decrease of reactive oxygen species under oxidative stress-inducing conditions.

[0028] The primary screening step uses a multi-well plate (e.g., a 96-well plate) as shown in the Examples below, and contacts a different candidate compound with plant cells in each well, thereby making it possible to rapidly screen a wide variety of compounds (e.g., several hundred compounds per day) while minimizing the amounts of plant cells and candidate compounds used. For example, in the Examples below, the amount of plant cells used in the primary screening step (amount after centrifugation) was only 25 mL, and the amount of each candidate compound was only 1 μL per procedure.

[0029] The reason for culturing under oxidative stress-inducing conditions in the primary screening step is to test the behavior of candidate compounds (whether they promote or suppress the production of reactive oxygen species) in a situation where plant cells are subjected to oxidative stress. The reason for culturing under oxidative stress-non-inducing conditions in the primary screening step is to eliminate compounds that are toxic to plant cells regardless of oxidative stress and compounds that inhibit the measurement of the amount of reactive oxygen species produced (i.e., compounds that are not suitable for the screening method according to the embodiment).

[0030] In the primary screening step, it is preferable to use two or more types of oxidative stress-inducing conditions. "Use of two or more types of oxidative stress-inducing conditions" means that at least both strong and weak oxidative stress-inducing conditions are set. When a substance such as paraquat described below is used, the strength of the oxidative stress-inducing conditions can be adjusted by increasing or decreasing the amount of the substance (high or low concentration).

[0031] The plant cells used in the screening step according to the embodiment are plant cells capable of photosynthesis. More specifically, the plant cells are T-87 strain green cultured cells derived from Arabidopsis thaliana. Hereinafter, the T-87 strain green cultured cells derived from Arabidopsis thaliana may also be referred to simply as "T-87 strain." The T-87 strain is used as a model for biochemical research due to the availability of abundant information on Arabidopsis thaliana and the availability of molecular and genetic tools. Furthermore, the T-87 strain maintains a photosynthetic system despite being a cultured cell, and retains the ability to produce reactive oxygen species derived not only from mitochondria but also from chloroplasts.

[0032] The substance used in the oxidative stress-inducing conditions in the screening step according to the embodiment is paraquat (methyl viologen, 1,1'-dimethyl-4,4'-bipyridinium dichloride). Paraquat is known as a component of non-selective herbicides, and generates reactive oxygen species when it acts on cells.

[0033] In the primary screening step, the amount of reactive oxygen species produced is measured using a chemiluminescent probe, specifically MCLA (methyl cypridina luciferin analog).

[0034] The secondary screening step is a step of contacting plant cells with primary candidate compounds, culturing them under oxidative stress-inducing conditions and under non-oxidative stress-inducing conditions, and measuring the viability of the plant cells to select secondary candidate compounds from the primary candidate compounds. The secondary screening step is a step performed to facilitate the elimination of compounds that do not affect cell death caused by oxidative stress and to increase the efficiency of screening.

[0035] The secondary screening is performed from the following two perspectives. The first perspective is whether the viability of plant cells after culture under oxidative stress-non-inducing conditions is significantly lower than the viability of plant cells in a control cultured under oxidative stress-non-inducing conditions without contacting the plant cells with the primary candidate compound. The second perspective is whether the viability of plant cells after culture under oxidative stress-inducing conditions is significantly lower or higher than the viability of plant cells in a control cultured under oxidative stress-inducing conditions without contacting the plant cells with the primary candidate compound. The secondary candidate compound can be said to be a compound that increases or decreases the viability of plant cells by affecting the increase or decrease of reactive oxygen species under oxidative stress-inducing conditions.

[0036] In the secondary screening step, a multi-well plate (e.g., a 96-well plate) as shown in the Examples below is used, and a different primary candidate compound is brought into contact with plant cells in each well, thereby making it possible to rapidly screen a wide variety of compounds while reducing the amounts of plant cells and primary candidate compounds used.

[0037] The reason for culturing under oxidative stress-inducing conditions in the secondary screening step is to test the behavior of candidate compounds (whether they decrease or increase the viability of plant cells) in a situation where plant cells are subjected to oxidative stress. The reason for culturing under oxidative stress-non-inducing conditions in the secondary screening step is to eliminate compounds that are toxic to plant cells regardless of oxidative stress and compounds that inhibit the measurement of plant cell viability (i.e., compounds that are not suitable for the screening method according to the embodiment).

[0038] In the secondary screening step, the viability of plant cells is measured by measuring autofluorescence derived from chlorophyll. Since the decrease in autofluorescence derived from chlorophyll is due to the breakdown of chloroplasts, the measurement of autofluorescence can be used to evaluate the viability of chloroplasts and, ultimately, plant cells.

[0039] If the secondary candidate compounds selected by the secondary screening process have the effect of increasing the viability of plant cells, they may be usable as components of biostimulants or disease prevention agents, and if they have the effect of decreasing the viability of plant cells, they may be usable as components of herbicides.

[0040] 2. Biostimulants, epidemic prevention agents, and herbicides according to embodiments The biostimulants, epidemic prevention agents, and herbicides according to embodiments include compounds selected by the screening methods according to the examples described below.

[0041] The biostimulant according to the embodiment contains the following compound (1). Compound (1) is known as [(E)-(1,3-dimethyl-5-oxopyrazol-4-ylidene)amino]4-chlorobenzoate. The biostimulant according to the embodiment may contain a substance other than compound (1).

[0042] The epidemic prevention agent according to the embodiment contains the following compound (2). The compound (2) is known as 4-(4-chlorophenyl)-6-methoxypyrimidin-2-amine. The epidemic prevention agent according to the embodiment may contain a substance other than compound (2).

[0043] The herbicide according to the embodiment contains the following compound (3). The name of compound (3) is 4-chloro-N-[2-chloro-4-(trifluoromethyl)phenyl]benzenesulfonamide. Note that the herbicide according to the embodiment may contain a substance other than compound (3).

[0044] 3. Effects of Screening Methods, Biostimulants, Epidemic Control Agents, and Herbicides According to Embodiments The screening methods according to embodiments include the above-described primary screening step and secondary screening step. Therefore, the screening methods according to embodiments can be performed using small amounts of plant cells and compounds, and can easily and quickly screen a wide variety of compounds, thereby reducing costs, time, and labor compared to conventional screening methods.

[0045] Furthermore, in the screening method according to the embodiment, the plant cells are photosynthetic plant cells, and in the secondary screening step, the viability of the plant cells is measured by measuring autofluorescence derived from chlorophyll. Therefore, the screening method according to the embodiment makes it possible to measure the viability of plant cells easily and in a short time.

[0046] Furthermore, in the screening method according to the embodiment, the plant cells are T-87 strain green cultured cells derived from Arabidopsis thaliana. Therefore, according to the screening method according to the embodiment, stable screening results can be obtained using the T-87 strain, which has relatively small cell masses, is easy to culture, and has stable photosynthetic ability.

[0047] Furthermore, in the screening method according to the embodiment, the substance used under the oxidative stress-inducing conditions is paraquat (methyl viologen), which acts on plant cells to stably generate reactive oxygen species, making it possible to obtain stable screening results.

[0048] Furthermore, in the screening method according to the embodiment, the amount of reactive oxygen species produced is measured using a chemiluminescent probe in the primary screening step, and therefore, the screening method according to the embodiment makes it possible to measure the amount of reactive oxygen species produced easily and in a short time.

[0049] In the screening method according to the embodiment, the chemiluminescent probe is MCLA (methyl cypridina luciferin analog), which has a stable structure and relatively long-lasting chemiluminescence, and thus the screening method according to the embodiment makes it possible to obtain stable screening results using MCLA.

[0050] Furthermore, the biostimulants, disease prevention agents, and herbicides according to the embodiments are useful drug candidates, including compounds for which new uses have been discovered by the screening methods according to the examples described below.

[0051] [Examples] In the examples described below, the screening method according to the embodiment was actually carried out, and compounds that could be candidates for biostimulants, disease prevention agents, and herbicides were selected from a plurality of candidate compounds.

[0052] 1. Preliminary Study Figure 1 is a graph showing the dose dependency of reactive oxygen species production and cell death when T-87 strain was cultured with paraquat in a preliminary study of the example. Figure 1(a) is a graph showing reactive oxygen species (ROS) production, and Figure 1(b) is a bar graph showing cell death. "MV" in Figure 1(b) refers to paraquat. In the figures described below, paraquat is also referred to as "MV." Note that "MV" comes from methyl viologen, another name for paraquat. The vertical axis of the graph in Figure 1(a) shows the chemiluminescence value (unit: rlu, relative luminescence units) derived from reactive oxygen species (ROS), and the horizontal axis shows the culture time (unit: h). In addition, in Figure 1(a), the symbols A, B, C, D, and E indicate graphs where the amount of paraquat added (final molar concentration) was 0 mM, 0.1 mM, 1 mM, 10 mM, and 20 mM, respectively. The chemiluminescence values ​​in Figure 1(a) are relative values, with the chemiluminescence value at 0 mM paraquat addition and 0 hours of incubation taken as 1. The vertical axis of the bar graph in Figure 1(b) indicates cell death (unit: %), and the horizontal axis indicates the amount of paraquat (MV) added (final molar concentration). In Figure 1(b), two bar graphs are lined up for each item of the amount of paraquat (MV) added (final molar concentration); the left bar graph is for 24 hours of incubation, and the right bar graph is for 48 hours of incubation. All data show the mean and standard error of 3 to 5 experiments, and significant differences were examined using Welch's t-test. P ＊ <0.05, P ＊＊ <0.01, P ＊＊＊ <0.001, P ＊＊＊＊ <0.0001.

[0053] First, a preliminary study on the culture of T-87 strain under oxidative stress-inducing conditions using paraquat was conducted to examine the dose-dependence of reactive oxygen species production and cell death, as well as the culture time. The amounts of paraquat added (final molar concentrations) in the preliminary study were 0 mM, 0.1 mM, 1 mM, 10 mM, and 20 mM. The culture times in the preliminary study were 6 hours, 15 hours, 24 hours, and 48 hours.

[0054] The T-87 strain, green cultured cells derived from Arabidopsis thaliana, which are plant cells used in the examples, were provided by the RIKEN BioResource Research Center (Ibaraki Prefecture). The T-87 strain was grown in a medium containing 3% sucrose and 200 mg / L of KH ２ P.O. ４ Liquid Murashige and Skoog (MS) medium (pH 5.7) supplemented with 0.2 mg / L thiamine hydrochloride, 100 mg / L myo-inositol, and 0.2 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) was used, and 100 mL of the liquid medium was placed in a 500 mL Erlenmeyer flask and grown at 22°C under continuous light conditions using a rotary shaker (100 rpm). Note that in the preliminary studies and screening methods according to the examples, cells on day 5 of culture were used.

[0055] The production of reactive oxygen species was measured by the following procedure (method using a chemiluminescent probe): First, 25 mL of cells were collected from the culture medium of the T-87 strain by centrifugation and then resuspended in 5 mM MES (2-(N-morpholino)ethanesulfonic acid), 0.5 mM CaCl ２ , 0.5 mM K ２ SO ４ The T-87 strain (100 μL) was resuspended in 120 mL of reactive oxygen species measurement buffer (pH 7.0) containing 175 mM mannitol. Using a multichannel pipette, 100 μL of the T-87 strain was dispensed into each well of a 96-well white plate for luminescence measurement (No. 236107, purchased from Thermo Fisher Scientific, USA; hereafter simply referred to as the "96-well plate"). After 1.5 hours of shaking, a specified amount of paraquat (purchased from Kanto Chemical Co., Ltd.) was added to each well to induce reactive oxygen species production. After incubation for the specified time, 10 μL of 10 mM MCLA (purchased from Kanto Chemical Co., Ltd.) dissolved in reactive oxygen species measurement buffer was added to each well, and reactive oxygen species-dependent chemiluminescence was recorded for 1 second using a luminometer (Ultra Evolution Microplate Reader, Tecan, Switzerland).

[0056] Cell death was measured using the following procedure (Evans Blue assay). First, 0.05% Evans Blue (purchased from Sigma-Aldrich, USA) was added to the cultured T-87 cell samples and incubated for 10 minutes. The cells were then washed four times with water to remove any unabsorbed dye. For each sample, more than 200 cells were counted using a bright-field microscope to determine cell viability.

[0057] As a result, it was confirmed that both reactive oxygen species production and cell death were dose-dependent with paraquat (see Figure 1). Based on the results of a preliminary study, the amount of paraquat added (final molar concentration) in the screening method according to the present example was set to 0.1 mM and 10 mM, the culture time in the primary screening step was set to 6 hours, and the culture time in the secondary screening step was set to 24 hours.

[0058] 2. Primary Screening Step Figure 2 shows the appearance of a 96-well plate used in the primary screening step and secondary screening step in the Examples. In Figure 2, "DMSO" indicates wells to which DMSO was added as a solvent control, and "Chemical Compounds" indicates the range of wells to which candidate compounds or primary candidate compounds were added. Figure 3 is a table showing an example of the results of the primary screening step in the Examples. Figure 3 is divided into three columns according to the amount of paraquat (MV) added, and the numbers at the top of each column and the letters at the left edge indicate the position of the wells on the 96-well plate (see Figure 2). The numerical values ​​(chemiluminescence values) at the locations corresponding to each well indicate the relative intensity of chemiluminescence (relative values ​​with the solvent control result set at 1).

[0059] As candidate compounds in the primary screening step, 9,991 types of low molecular weight compounds contained in a commercially available chemical library, DIVERSet NovaCore NQ612, 5 mg / mL DMSO (purchased from Thermo Fisher Scientific Inc. (USA)), were used.

[0060] The primary screening step was carried out by the following procedure (method using a chemiluminescent probe): First, 25 mL of cells were collected from the culture medium of the T-87 strain by centrifugation, and the cells were diluted with 5 mM MES, 0.5 mM CaCl ２ , 0.5 mM K ２ SO ４ The cells were resuspended in 120 mL of reactive oxygen species measurement buffer (pH 7.0) containing 175 mM mannitol. Buffer solution (100 μL) containing T-87 strain was dispensed into each well of a 96-well plate using a multichannel pipette. Then, 1 μL of a different candidate compound was added to each well (A2-H11) (final concentration: 25 μg / mL). In addition, DMSO (1%) was added to wells A1 and A12 located at both ends of the 96-well plate as a solvent control (see Figure 2). After shaking for 1.5 hours, a specified amount of paraquat (0.1 mM or 10 mM) was added to each well to induce reactive oxygen species production. Samples without paraquat were also prepared for comparison, using cultures under conditions that did not induce oxidative stress. After incubation for 6 hours, 10 μL of 10 mM MCLA dissolved in the reactive oxygen species measurement buffer was added to each well, and reactive oxygen species-dependent chemiluminescence was recorded for 1 second using a luminometer. The above procedure was performed in duplicate, and the average of the results was used for evaluation.

[0061] The primary screening step is a step in which plant cells are contacted with candidate compounds, then cultured under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, and the amount of reactive oxygen species produced is measured to perform primary screening and select primary candidate compounds from the candidate compounds. The selection method is as follows.

[0062] First, compounds (e.g., compounds C2, D2, E2, E4, E9, F7, G6, and H6 in Figure 3 ) that were confirmed to have significantly lower chemiluminescence values ​​than the solvent control (DMSO only) when cultured under non-oxidative stress-inducing conditions (i.e., 0 mM paraquat) were excluded because they were considered to be directly toxic to cells or to inhibit chemiluminescence itself. The criteria for whether the chemiluminescence value obtained with the addition of a candidate compound is significantly lower than the chemiluminescence value obtained with the solvent control depend on the desired screening accuracy, but one guideline is whether the chemiluminescence value obtained with the addition of a candidate compound is less than 50% of the chemiluminescence value obtained with the solvent control. The chemiluminescence value obtained with the solvent control was the average of the chemiluminescence values ​​obtained with wells A1 and A12.

[0063] Next, from among the candidate compounds excluding compounds eliminated by the above criteria, compounds that significantly affect the production of reactive oxygen species induced by the addition of paraquat were selected. That is, compounds confirmed to have chemiluminescence values ​​significantly lower than the solvent control results (e.g., the compounds in wells A3 and C8 in Figure 3) and compounds confirmed to have chemiluminescence values ​​significantly higher than the solvent control results (e.g., the compounds in wells A6 and H8 in Figure 3) were selected and used as primary candidate compounds. The criteria for whether the chemiluminescence value with the addition of a candidate compound is significantly lower or higher than the solvent control chemiluminescence value will depend on the desired screening accuracy, etc., but can be, for example, whether the chemiluminescence value is lower than 50% and higher than 150% of the solvent control chemiluminescence value, respectively.

[0064] 3. Secondary Screening Step Figure 4 is a table showing an example of the results of the secondary screening step in the examples. Figure 4 is divided into two sections based on the amount of paraquat added, and the numbers at the top of each section and the letters at the left edge indicate the position of the wells in the 96-well plate (see Figure 2). The numerical values ​​(autofluorescence values) at each well indicate the relative intensity of autofluorescence derived from chlorophyll (relative values ​​with the solvent control result set at 1). Note that the well positions in Figure 4 are indicated in the same way as in Figure 3, but the compounds added to wells A2 to H11 in the example in Figure 4 are different from the compounds added to wells A2 to H11 in the example in Figure 3.

[0065] The secondary screening process (method for measuring chlorophyll-derived autofluorescence) was performed as follows. First, 25 mL of cells were collected from the T-87 strain culture by centrifugation and resuspended in 120 mL of reactive oxygen species measurement buffer. The buffer solution containing the T-87 strain (100 μL) was dispensed into each well of a 96-well plate using a multichannel pipette. Then, 1 μL of a primary candidate compound selected from the candidate compounds was added to each well (A2-H11) (final concentration: 25 μg / mL). In addition, DMSO (1%) was added as a solvent control to wells A1 and A12 located at both ends of the 96-well plate (see Figure 2). After shaking for 1.5 hours, a specified amount of paraquat (10 mM) was added to each well to induce chloroplast dysfunction. For comparison, a sample without paraquat was also prepared for culture under conditions that did not induce oxidative stress. After 24 hours of incubation, the chloroplast autofluorescence was recorded for 0.1 seconds using a luminometer (excitation wavelength: 480 nm, emission wavelength: 740 nm). The above procedure was carried out in duplicate, and the average values ​​were used for evaluation.

[0066] The secondary screening step is a step of contacting plant cells with primary candidate compounds, culturing them under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, and measuring the viability of the plant cells to perform secondary screening, thereby selecting secondary candidate compounds from the primary candidate compounds. The selection method is as follows.

[0067] First, compounds that were confirmed to have significantly lower autofluorescence values ​​than the solvent control (DMSO only) when cultured under non-oxidative stress-inducing conditions (i.e., 0 mM paraquat) (not applicable in the results illustrated in Figure 4) were excluded because they were considered to be directly toxic to cells or to inhibit autofluorescence itself. The criteria for whether the autofluorescence value when the primary candidate compound was added was significantly lower than the autofluorescence value in the solvent control depend on factors such as the desired screening accuracy, but one guideline can be, for example, whether the autofluorescence value is less than 50% of the autofluorescence value in the solvent control. The autofluorescence value in the solvent control was taken as the average of the autofluorescence values ​​in wells A1 and A12.

[0068] Next, from the primary candidate compounds, excluding compounds eliminated by the above criteria, compounds that significantly affected chloroplast decay (i.e., plant cell viability) induced by the addition of paraquat were selected. That is, compounds for which autofluorescence was confirmed to be significantly lower than that of the solvent control (e.g., compounds in wells F4, F5, and F11 in Figure 4 ) and compounds for which autofluorescence was confirmed to be significantly higher (e.g., compounds in wells A8, B5, C7, C8, E7, and H6 in Figure 4 ) were selected as secondary candidate compounds. The criteria for whether the autofluorescence value after addition of the primary candidate compound is significantly lower or higher than that of the solvent control depend on the desired screening accuracy, but can be, for example, whether the autofluorescence value is lower or higher than that of the solvent control, respectively. The secondary candidate compounds in the examples included the following compounds (1), (2), and (3).

[0069] 4. Test using Arabidopsis thaliana plants Finally, it was confirmed whether the compounds screened by the screening method of the present example can be used as biostimulants, disease prevention agents, or herbicides using Arabidopsis thaliana plants. Compounds (1), (2), and (3) for the test using Arabidopsis thaliana plants were obtained from Namiki Shoji Co., Ltd.

[0070] 4-1. Biostimulant Figure 5 is a diagram illustrating the effect of compound (1) as a biostimulant. Figure 5(a) is a schematic diagram of a test conducted to confirm the effect of compound (1) as a biostimulant, and Figure 5(b) is a photograph showing the test results.

[0071] The secondary candidate compound, Compound (1), was tested for its function as a biostimulant (see Figure 5(a)). First, Arabidopsis thaliana (Col-0, hereinafter the same) 1 week after germination were grown on ½ MS agar medium containing Compound (1) (agar-solidified medium with half the concentration of inorganic salts in MS medium, pH 5.7) under long-day conditions (16 hours light / 8 hours dark, 22°C) for 5 days (chemical treatment). The Arabidopsis thaliana was then transferred to ½ MS agar medium containing paraquat (MV) (MV treatment) and grown under long-day conditions for 4 days, after which the condition was visually confirmed (visible selection). A control test was also conducted using DMSO instead of Compound (1). The molar concentrations of Compound (1) and paraquat on ½ MS agar medium were both 50 μM.

[0072] As a result, it was confirmed that Arabidopsis thaliana plants turned white and died in the control test using DMSO, whereas in the test using compound (1), it was confirmed that Arabidopsis thaliana plants maintained their green color and continued to grow (see Figure 5(b)).

[0073] 6 is a bar graph showing the effect of compound (2) as a plant disease preventive agent. The vertical axis of the bar graph in Fig. 6 shows the bacterial density (colony forming units per mg of plant weight) (unit: CFU / mg), and the horizontal axis shows the compound added.

[0074] The secondary candidate compound, Compound (2), was tested for its function as a disease prevention agent. In this test, P. syringae pv. tomato DC3000 (hereinafter referred to as DC3000 strain) was used as the plant pathogenic bacterium. The DC3000 strain was cultured at 28°C for 24 hours on mannitol-glutamic acid (MG) agar medium containing rifampicin (50 μg / mL). Arabidopsis thaliana was grown for 10 days under long-day conditions on ½ MS agar medium containing Compound (2). The molar concentration of Compound (2) on the ½ MS agar medium was 25 μM.

[0075] Arabidopsis root tips were incubated in a diluted bacterial culture medium (OD ６００ The DC3000 strain was inoculated by immersion in 5% H2O2 (pH 7.0:0.002) for 1 second. After inoculation, Arabidopsis plants were transferred to new 1 / 2 MS agar medium and grown under long-day conditions for 7 days. To examine the growth of DC3000 on Arabidopsis, the plants were surface-sterilized by immersion in 5% H2O2 for 2 minutes. After washing three times with sterile water, six Arabidopsis samples were homogenized with 5 mL of sterile water using a mortar and pestle. Appropriately diluted samples were then plated on MG agar medium. After incubation at 30°C for several days, the colonies formed on the plates were counted, and the bacterial density was expressed as colony-forming units per mg of plant weight. In addition, tests were also carried out using DMSO as a control and BTH (Acibenzolar-S-methyl) (purchased from Fujifilm Wako Pure Chemical Industries, Ltd.), a conventionally known antimicrobial agent, instead of compound (2).

[0076] As a result, it was confirmed that compound (2) clearly inhibited the proliferation of DC3000 strain compared to the case where DMSO was used (control) (see Figure 6). All data show the mean and standard error of 3 to 5 experiments, and significance was examined by Welch's t-test. ＊＊＊ <0.001.

[0077] 4-3. Herbicide Figure 7 is a diagram illustrating the herbicide effect of compound (3). Figure 7(a) is a schematic diagram showing the test conducted to confirm the herbicide effect of compound (3), Figure 7(b) is a photograph showing the test results for DMSO, and Figure 7(c) is a photograph showing the test results for compound (3).

[0078] The secondary candidate compound, compound (3), was tested for its function as a herbicide (see FIG. 7(a)). First, Arabidopsis thaliana plants 1 week after germination were cultivated on ½ MS agar medium (pH 5.7) containing compound (3) under long-day conditions (16 hours light / 8 hours dark, 22°C) for 5 days (chemical treatment). A control test was also conducted using DMSO instead of compound (3). Thereafter, the state of Arabidopsis thaliana plants was confirmed by visual observation (visible selection). The molar concentration of compound (3) on ½ MS agar medium was set to 50 μM.

[0079] As a result, it was confirmed that in the control test using DMSO, Arabidopsis thaliana remained green and grew normally (see Figure 7(b)). On the other hand, in the test using compound (3), it was confirmed that Arabidopsis thaliana turned white and experienced growth abnormalities or withering (see Figure 7(c)).

[0080] 5. Results The above test examples confirmed that the screening method of the present invention can be carried out using small amounts of plant cells and compounds, and is capable of screening a wide variety of compounds simply and quickly, thereby reducing costs, time, and labor compared to conventional screening methods.

[0081] Furthermore, the above test examples confirmed that the biostimulants, disease prevention agents and herbicides of the present invention are useful drug candidates, including compounds for which new uses have been discovered by the screening method of the present invention.

[0082] Although the present invention has been described above based on the above embodiment, the present invention is not limited to the above embodiment and can be embodied in various forms without departing from the spirit of the present invention, and for example, the following modifications are also possible.

[0083] (1) In the above embodiment, the plant cells are photosynthetic plant cells, and the viability of the plant cells is measured by measuring chlorophyll-derived autofluorescence in the secondary screening step, but the present invention is not limited to this. If a method for measuring chlorophyll-derived autofluorescence is not used in the secondary screening step, the plant cells may be non-photosynthetic plant cells. In this case, the viability of the plant cells can be measured in the secondary screening step, for example, by Evans Blue assay.

[0084] (2) In the above embodiment, the plant cells are Arabidopsis thaliana-derived green cultured cell line T-87, but the present invention is not limited thereto. Other plant cells (e.g., tobacco-derived green cultured cell line NI) may also be used as photosynthetic plant cells.

[0085] (3) In the above embodiment, the substance used under the oxidative stress-inducing conditions is paraquat (methyl viologen), but the present invention is not limited to this. Other oxidative stress-inducing substances may also be used.

[0086] (4) In the above embodiment, the amount of reactive oxygen species produced is measured using a chemiluminescent probe in the primary screening step, but the present invention is not limited to this. The amount of reactive oxygen species produced may also be measured by other methods (e.g., measurement using fluorescence or a redox reaction).

[0087] (5) In the above embodiment, the chemiluminescent probe is MCLA, but the present invention is not limited to this. Other chemiluminescent probes (e.g., CLA-related reagents such as CLA, FCLA, and Red-CLA) may also be used.

Claims

1. A screening method for evaluating whether a plurality of candidate compounds can be used as a plant biostimulant, a plant disease prevention agent, or a plant herbicide, comprising: a primary screening step of contacting plant cells with the candidate compounds, culturing the cells under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, measuring the amount of reactive oxygen species produced, and selecting a primary candidate compound from the candidate compounds; a secondary screening step of contacting the plant cells with the primary candidate compounds, then culturing them under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, and measuring the viability of the plant cells to perform secondary screening, thereby selecting secondary candidate compounds from the primary candidate compounds.

2. The plant cells are photosynthetic plant cells, 2. The screening method according to claim 1, wherein the viability of the plant cells is determined by measuring autofluorescence derived from chlorophyll in the secondary screening step.

3. 3. The method of claim 2, wherein the plant cells are T-87 strain green cultured cells derived from Arabidopsis thaliana.

4. 2. The screening method according to claim 1, wherein the substance used under the oxidative stress-inducing conditions is paraquat (methyl viologen).

5. 2. The screening method according to claim 1, wherein the amount of reactive oxygen species produced is measured using a chemiluminescent probe in the primary screening step.

6. 6. The screening method according to claim 5, wherein the chemiluminescent probe is MCLA (methyl cypridina luciferin analog).