JP7813083B2 - Screening Method - Google Patents

Description

Cross Reference

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

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 .

Traditionally, there has been extensive research into compounds that can be used as active ingredients in plant-affecting agents (biostimulants, disease control agents, or herbicides). However, in recent years, the number of compounds required to develop a single product (i.e., the number of compounds that must be screened) has increased significantly, resulting in a significant decline in the frequency of drug development.

For example, in conventional screening methods for disease prevention agents, candidate compounds for disease prevention agents are administered to actual plants such as rice along with pathogens, and visual selection is carried out using the spread of disease spots 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 the compound, is also commonly used in screening methods for herbicides.

JP 2009-72070 A

However, conventional screening methods for biostimulants, disease control agents or herbicides have the disadvantage of requiring significant expense, time and effort.

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 that contain compounds selected by the screening method of the present invention.

[1] The screening method of the present invention is a screening method for evaluating whether multiple candidate compounds can be used as plant biostimulants, disease prevention agents, or herbicides, and is characterized by comprising a primary screening step in which plant cells are contacted with the 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 a secondary screening step in which the plant cells are contacted with the primary candidate compounds, then cultured under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, and the viability of the plant cells is measured to perform secondary screening, and a secondary candidate compound is selected from the primary candidate compounds.

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

[3] In the screening method described in [2] above, it is preferable that the plant cells are green cultured cells of the T-87 strain derived from Arabidopsis thaliana.

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

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

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

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

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

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

The screening method of the present invention includes a primary screening 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 a primary screening and select primary candidate compounds from the candidate compounds; and a secondary screening step in which plant cells are contacted with the primary candidate compounds, then cultured under oxidative stress-inducing conditions and under oxidative stress-non-inducing conditions, and the viability of the plant cells is measured to perform a secondary screening and select 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, resulting in a screening method that reduces cost, time, and labor compared to conventional screening methods.

In addition, the biostimulants, disease prevention agents, and herbicides of the present invention are useful drug candidates, including compounds for which new uses have been discovered through the screening methods of the present invention.

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

The screening method, biostimulant, disease prevention agent, and herbicide of the present invention are described below based on embodiments.

[Embodiment]
1. Screening Method According to an Embodiment The screening method according to an embodiment 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 an embodiment includes a primary screening step and a secondary screening step.

In this specification, 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 (e.g., stress resulting from temperature, ultraviolet light, physical stimuli, etc.) (eliminating or suppressing the effects of various abiotic stresses).

In addition, the term "disease control agent" as used herein refers to an agent that has the effect of protecting plants from at least one pathogen (preventing or suppressing the effects of pathogens).

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 part of a plant.

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

As used herein, "oxidative stress-inducing conditions" refer to conditions that favor the generation of reactive oxygen species in plant cells. Reactive oxygen species (ROS) is a collective term for highly reactive substances 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 not only have direct antibacterial effects but also play multiple beneficial roles within cells, such as signaling molecules that regulate the expression of stress-responsive resistance genes and various metabolic pathways. On the other hand, at high concentrations, reactive oxygen species 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).

Primary screening is performed from the following two perspectives. The first perspective is whether the amount of reactive oxygen species produced after culture under non-oxidative stress-inducing conditions is significantly less than the amount of reactive oxygen species produced in a control where plant cells are cultured under non-oxidative stress-inducing conditions without contacting them with the 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 where plant cells are cultured under oxidative stress-inducing conditions without contacting them with the candidate compound. Primary candidate compounds can be said to be reactive oxygen regulators that affect the increase or decrease of reactive oxygen species under oxidative stress-inducing conditions.

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

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 situations where plant cells are subjected to oxidative stress. The reason for culturing under non-oxidative stress-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 of the embodiment).

In the primary screening process, it is preferable to use two or more types of oxidative stress-inducing conditions. "Use two or more types of oxidative stress-inducing conditions" means setting at least both strong and weak oxidative stress-inducing conditions. When using a substance such as paraquat, which will be described later, the strength of the oxidative stress-inducing conditions can be adjusted by adjusting the amount added (high or low concentration).

The plant cells used in the screening process according to the embodiment are photosynthetic plant cells. More specifically, the plant cells are T-87 strain green cultured cells derived from Arabidopsis thaliana. The T-87 strain green cultured cells derived from Arabidopsis thaliana may hereinafter 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, despite being a cultured cell, the T-87 strain maintains a photosynthetic system and retains the ability to produce reactive oxygen species derived not only from mitochondria but also from chloroplasts.

The substance used in the oxidative stress-inducing conditions in the screening process according to this 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.

In the primary screening process, the amount of reactive oxygen species produced is measured using a chemiluminescent probe. Specifically, the chemiluminescent probe is MCLA (methyl cypridina luciferin analog).

The secondary screening process involves contacting plant cells with primary candidate compounds, culturing them under oxidative stress-inducing conditions and non-oxidative stress-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 secondary screening process is carried out to facilitate the elimination of compounds that do not affect cell death caused by oxidative stress and to increase the efficiency of screening.

Secondary screening is performed from the following two perspectives. The first perspective is whether the viability of plant cells after culture under non-oxidative stress-inducing conditions is significantly lower than the viability of plant cells in a control cultured under non-oxidative stress-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. Secondary candidate compounds can be said to be compounds that increase or decrease the viability of plant cells by affecting the increase or decrease of reactive oxygen species under oxidative stress-inducing conditions.

In the secondary screening process, 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 contacted with plant cells in each well, making it possible to rapidly screen a wide variety of compounds while minimizing the amount of plant cells and primary candidate compounds used.

The reason for culturing under oxidative stress-inducing conditions in the secondary screening step is to test the behavior of candidate compounds (whether they reduce or increase the viability of plant cells) in situations where plant cells are subjected to oxidative stress. The reason for culturing under non-oxidative stress-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 of the embodiment).

In the secondary screening process, the viability of plant cells is measured by measuring autofluorescence derived from chlorophyll. Because the decrease in chlorophyll-derived autofluorescence is due to the breakdown of chloroplasts, measuring this autofluorescence can be used to evaluate the viability of chloroplasts and, ultimately, plant cells.

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

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.

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).

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).

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).

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.

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.

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.

In addition, in the screening method according to the embodiment, the substance used under the oxidative stress-inducing conditions is paraquat (methyl viologen). Therefore, according to the screening method according to the embodiment, stable screening results can be obtained using paraquat, which acts on plant cells to stably generate reactive oxygen species.

In addition, 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. 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.

In addition, in the screening method according to the embodiment, the chemiluminescent probe is MCLA (methyl cypridina luciferin analog). Therefore, according to the screening method according to the embodiment, stable screening results can be obtained using MCLA, which has a stable structure and relatively long-lasting chemiluminescence.

In addition, the biostimulants, disease prevention agents, and herbicides of the embodiments are useful drug candidates, including compounds for which new uses have been discovered using the screening methods of the examples described below.

[Example]
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.

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 Figure 1(a), the symbols A, B, C, D, and E represent graphs in which 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 and 0 hours of incubation taken as 1. The vertical axis of the bar graph in Figure 1(b) represents cell death (unit: %), and the horizontal axis represents the amount of paraquat (MV) added (final molar concentration). In Figure 1(b), two bar graphs are lined up for each amount of paraquat (MV) added (final molar concentration); the left bar graph represents the 24-hour incubation, and the right bar graph represents the 48-hour incubation. All data represent the mean and standard error of three to five experiments, and significance was examined using Welch's t-test. P ^* <0.05, P ^** <0.01, P ^*** <0.001, P ^*** <0.0001.

First, in a preliminary study on culturing the T-87 strain under oxidative stress-inducing conditions using paraquat, we investigated the dose dependency of reactive oxygen species production and cell death as well as the incubation 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 incubation times in the preliminary study were 6 hours, 15 hours, 24 hours, and 48 hours.

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

Reactive oxygen species production was measured using the following procedure (a method using a chemiluminescent probe). First, 25 mL of cells were collected from the culture medium of the T-87 strain by centrifugation and resuspended in 120 mL of reactive oxygen species measurement buffer (pH 7.0) containing 5 mM MES (2-(N-morpholino)ethanesulfonic acid), _0.5 mM CaCl , _0.5 mM _K SO , and 175 mM mannitol. T-87 strain (100 μL) 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") using a multichannel pipette. After shaking for 1.5 hours, a specified amount of paraquat (purchased from Kanto Chemical Co., Inc.) was added to each well to induce reactive oxygen species production. After incubation for a predetermined time, 10 μL of 10 mM MCLA (purchased from Kanto Chemical Co., Ltd.) dissolved in a buffer solution for reactive oxygen species measurement was added to each well, and reactive oxygen species-dependent chemiluminescence was recorded for 1 second using a luminometer (Ultra Evolution Microplate Reader from Tecan, Switzerland).

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.

As a result, a dose dependency of paraquat was confirmed for both reactive oxygen species production and cell death (see Figure 1). Based on the results of preliminary studies, the amounts of paraquat added (final molar concentrations) in the screening method of the present example were set to 0.1 mM and 10 mM, the incubation time in the primary screening step was set to 6 hours, and the incubation time in the secondary screening step was set to 24 hours.

2. Primary Screening Step Figure 2 shows the appearance of a 96-well plate used in the primary screening step and the 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 in the 96-well plate (see Figure 2). The numerical value (chemiluminescence value) at the position corresponding to each well indicates the relative intensity of chemiluminescence (a relative value where the result for the solvent control is set to 1).

The candidate compounds used in the primary screening process were 9,991 small molecule compounds contained in the commercially available chemical library DIVERSet NovaCore NQ612, 5 mg/mL DMSO (purchased from Thermo Fisher Scientific, USA).

The primary screening process was performed using the following procedure (a method using a chemiluminescent probe). First, 25 mL of cells were collected from the culture medium of the T-87 strain by centrifugation and resuspended in 120 mL of a buffer solution for measuring reactive oxygen species (pH 7.0) containing ₅ mM MES, 0.5 mM CaCl , 0.5 mM _K SO , _and 175 mM mannitol. The buffer solution (100 μL) containing the 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 to H11) (final concentration: 25 μg/mL). Furthermore, 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 (0.1 mM or 10 mM) was added to each well to induce reactive oxygen species production. For comparison, samples without paraquat were also prepared. After culturing each sample 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.

The primary screening process involves contacting plant cells with candidate compounds, culturing them under oxidative stress-inducing conditions and under non-oxidative stress-inducing conditions, measuring the amount of reactive oxygen species produced, and selecting primary candidate compounds from the candidate compounds. The selection method is as follows:

First, compounds (e.g., compounds C2, D2, E2, E4, E9, F7, G6, and H6 in Figure 3) that were found 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 are considered to be directly toxic to cells or to inhibit chemiluminescence itself. The criteria for whether the chemiluminescence value when a candidate compound is added is significantly lower than the chemiluminescence value in the solvent control depend on factors such as the desired screening accuracy, but one guideline is whether the chemiluminescence value is below 50% of the chemiluminescence value in the solvent control. The chemiluminescence value in the solvent control was calculated as the average of the chemiluminescence values in wells A1 and A12.

Next, from among the candidate compounds, excluding those eliminated by the above criteria, compounds that significantly affected the production of reactive oxygen species induced by the addition of paraquat were selected. That is, compounds with chemiluminescence values significantly lower than the solvent control results (e.g., the compounds in wells A3 and C8 in Figure 3) and compounds with chemiluminescence values significantly higher than the solvent control results (e.g., the compounds in wells A6 and H8 in Figure 3) were selected and designated as primary candidate compounds. The criteria for whether the chemiluminescence values with the addition of a candidate compound are significantly lower or higher than the solvent control chemiluminescence values depend on factors such as the desired screening accuracy, but can be, for example, whether the chemiluminescence values are lower than 50% and higher than 150% of the solvent control chemiluminescence values, respectively.

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.

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 containing 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 1.5 hours of shaking, a specified amount of paraquat (10 mM) was added to each well to induce chloroplast dysfunction. Samples without paraquat were also prepared for comparison, with cultures 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.

The secondary screening process involves 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 perform secondary screening, thereby selecting secondary candidate compounds from the primary candidate compounds. The selection method is as follows.

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 shown in Figure 4) were excluded because they are considered to be directly toxic to cells or to inhibit autofluorescence itself. The criteria for whether the autofluorescence value when a primary candidate compound is added is significantly lower than that of the solvent control depend on factors such as the desired screening accuracy, but one guideline is whether the autofluorescence value is below 50% of the autofluorescence value of the solvent control. The autofluorescence value of the solvent control was taken as the average of the autofluorescence values of wells A1 and A12.

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).

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.

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.

The secondary candidate compound, Compound (1), was tested for its function as a biostimulant (see Figure 5(a)). First, Arabidopsis thaliana (Col-0, hereafter referred to as "Col-0"), 1 week after germination, was grown on 1/2 MS agar medium containing Compound (1) (MS medium with half the concentration of inorganic salts solidified with agar, 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 1/2 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 experiment was also conducted using DMSO instead of Compound (1). The molar concentrations of compound (1) and paraquat in the 1/2 MS agar medium were both 50 μM.

As a result, it was confirmed that in the control test using DMSO, Arabidopsis thaliana turned white and died. On the other hand, in the test using compound (1), it was confirmed that Arabidopsis thaliana remained green and continued to grow (see Figure 5(b)).

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.

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 under long-day conditions for 10 days on 1/2 MS agar medium containing compound (2). The molar concentration of compound (2) on the 1/2 MS agar medium was 25 μM.

Arabidopsis root tips were inoculated with the DC3000 strain by dipping them into a diluted bacterial culture medium (OD ₆₀₀ : 0.002) for 1 second. After inoculation, Arabidopsis plants were transferred to new 1/2 MS agar plates and grown under long-day conditions for 7 days. To examine the growth of DC3000 on Arabidopsis, the plants were surface-sterilized by immersing them 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 plates. 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).

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

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).

The secondary candidate compound, compound (3), was tested for its ability to function as a herbicide (see Figure 7(a)). First, Arabidopsis thaliana plants were grown one week after germination on 1/2 MS agar medium (pH 5.7) containing compound (3) under long-day conditions (16 hours light/8 hours dark, 22°C) for five days (chemical treatment). A control test was also conducted using DMSO instead of compound (3). The condition of the Arabidopsis thaliana plants was then visually confirmed (visible selection). The molar concentration of compound (3) on the 1/2 MS agar medium was set to 50 μM.

As a result, in the control test using DMSO, it was confirmed that 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)).

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.

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 using the screening method of the present invention.

The present invention has been described above based on the above embodiment, but the present invention is not limited to the above embodiment. It can be implemented in various ways without departing from the spirit of the invention, and for example, the following modifications are also possible:

(1) In the above embodiment, the plant cells are photosynthetic plant cells, and the viability of the plant cells is measured in the secondary screening step by measuring chlorophyll-derived autofluorescence, 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 plant cells that do not have photosynthetic ability. In this case, the viability of the plant cells can be measured in the secondary screening step, for example, by Evans Blue assay.

(2) In the above embodiment, the plant cells are the T-87 strain of green cultured cells derived from Arabidopsis thaliana, but the present invention is not limited to this. Other plant cells (e.g., the NI strain of green cultured cells derived from tobacco) may also be used as plant cells capable of photosynthesis.

(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.

(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 using other methods (e.g., measurement using fluorescence or a redox reaction).

(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 (6)

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. 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. The screening method described in claim 2, characterized in that the plant cells are T-87 strain green cultured cells derived from Arabidopsis thaliana. 2. The screening method according to claim 1 , wherein the substance used under the oxidative stress-inducing conditions is paraquat (methyl viologen). 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. The screening method of claim 5, wherein the chemiluminescent probe is MCLA (methyl cypridina luciferin analog).