JP2024054094A - Agent for inhibiting plant fungal diseases for use in treatment at the soil edge, method for inhibiting plant fungal diseases, agent for promoting expression of plant leaf signal transduction-related genes for use in treatment at the soil edge, method for promoting expression of plant leaf signal transduction-related genes, agent for promoting expression of plant main root signal transduction-related genes for use in treatment at the soil edge, and method for promoting expression of plant main root signal transduction-related genes - Google Patents

Abstract

[Problem] The objective of the present invention is to provide a disease inhibitor or a signal transduction-related gene expression promoter that is highly safe, has little phytotoxicity, is easy to apply, etc., and exhibits an excellent plant disease inhibitory effect or an associated gene expression effect that inhibits plant diseases.Solution: A plant fungal disease inhibitor for treatment at the ground edge, a signal transduction-related gene expression promoter in plant leaves for treatment at the ground edge, or a signal transduction-related gene expression promoter in plant main roots for treatment at the ground edge, each of which contains acetic acid as an active ingredient.[Selected Figure] None

Description

The present invention relates to a fungal disease inhibitor for use at the ground edge, a plant leaf signal transduction-related gene expression promoter for use at the ground edge, and a plant main root signal transduction-related gene expression promoter for use at the ground edge. In particular, the present invention relates to a fungal disease inhibitor for use at the ground edge, a plant leaf signal transduction-related gene expression promoter, and a plant main root signal transduction-related gene expression promoter for use at the ground edge, which contain acetic acid as an active ingredient.

Synthetic pesticides have traditionally been widely used as disease inhibitors (fungicides) for agricultural and horticultural crops. Many of these synthetic pesticides have a high disease inhibitory effect when used appropriately for the target disease.

However, synthetic pesticides can also cause problems with environmental pollution, so there is a growing demand for disease inhibitors that do not pollute the surrounding environment, are highly safe for humans, and have excellent disease suppression properties.

Therefore, disease inhibitors containing substances derived from natural products as active ingredients have come to be used. Natural substances that are also used as food raw material ingredients include fatty acid glycerides (Patent Document 1, etc.).
However, in order for these substances to become widely used as water-borne disease inhibitors (water-borne fungicides) for horticulture, there was a problem in that blending techniques were required, including combinations with other compounds and blending conditions.

On the other hand, from the viewpoint of solubility in water, acetic acid (vinegar, etc.) has been used (Patent Document 2, etc.). However, by adding vinegar to the hot water used in the germination treatment in which the rice seeds are soaked in hot water at a specified temperature for a specified time to germinate the rice seeds, bacteria and fungi latent in the rice seeds are secondarily killed or inactivated during the germination treatment, which is not a simple treatment and cannot be applied after planting. For this reason, there has been a demand for the development of a disease inhibitor containing acetic acid that is highly safe for humans and can be applied by users by spraying it on planted plants.

When diseases occur on stems and leaves, the common method of treatment is to spray pesticides on the affected areas. However, stems and leaves have a large surface area, and their shapes are complex and intricate, and the sprayed pesticides tend to flow in a direction different from the intended direction due to wind, etc., making it difficult to spray the pesticide evenly and sufficiently, including on the front and back of the leaves. As a result, this is a tedious task for users, and there is also the problem that the actual disease suppression effect is lower than the theoretical disease suppression effect.

Furthermore, genes related to salicylic acid signaling and jasmonic acid signaling are known as genes related to suppressing plant diseases. When disease occurs in stems and leaves, it was thought that the expression of the disease suppression-related genes would be controlled by spraying agrochemicals or the like on the affected area. However, when agrochemicals or the like are sprayed on stems and leaves for treatment, as with disease control agents, the surface area of the stems and leaves is large, and the shape is complex and three-dimensionally intricate. In addition, the sprayed agrochemicals tend to flow in a direction different from the intended direction due to wind or the like, so it is difficult to spray evenly and sufficiently, including the front and back of the leaves. As a result, it is a tedious task for the user, and there is also a problem that the actual expression levels of salicylic acid signaling and jasmonic acid signaling-related genes are lower than the theoretical expression levels. In other words, there is also a problem that the actual disease suppression effect is lower than the theoretical disease suppression effect.

JP 2005-170892 A JP 2006-50982 A

Sato, I., et al., Microbes Environ. Vol. 29, No. 2, 168-177, 2014 Takahashi, H., et al., Plant Cell Rep (2014) 33:99-110

The present invention aims to provide a disease suppressor or a signal transduction-related gene expression promoter that is highly safe, has little phytotoxicity, is easy to apply, and exhibits excellent plant disease suppression effects or related gene expression effects that suppress plant disease.

As a result of intensive research conducted in light of the above circumstances, the present inventors have discovered a plant disease inhibitor and a signal transduction-related gene expression promoter that contain acetic acid as an active ingredient.

That is, the agent for suppressing plant fungal diseases for use in treating soil edge of the present invention is characterized by containing acetic acid as an active ingredient.

The fungal disease may be powdery mildew.

The plant may be a rose or a cucumber.

The acidity of the acetic acid is preferably 0.05% or more and 2.0% or less.

The method for suppressing a fungal disease in a plant of the present invention includes a step of treating the ground edge of the plant with the above-mentioned fungal disease suppressant for treating the ground edge.

The plant leaf signal transduction-related gene expression promoter for use in soil edge treatment of the present invention is characterized by containing acetic acid as an active ingredient.

The signal transduction-related gene is preferably PR1, P4 or PR3.

The acidity of the acetic acid is preferably 0.05% or more and 2.0% or less.

The plant may be a tomato.

The method of promoting leaf signaling-related gene expression in a plant of the present invention includes a step of treating the ground edge of the plant with the plant leaf signaling-related gene expression promoter for treatment of the ground edge.

The plant main root signal transduction-related gene expression promoter for use in treating the soil edge of the plant according to the present invention is characterized by containing acetic acid as an active ingredient.

The signal transduction-related gene is preferably PR1 or P4.

The acidity of the acetic acid is preferably 0.05% or more and 2.0% or less.

The plant may be a tomato.

The method of promoting the expression of a gene related to signal transduction in the main root of a plant of the present invention includes a step of treating the ground edge of the plant with the above-mentioned plant main root signal transduction-related gene expression promoter for treating the ground edge.

The present invention can provide a disease suppressor or a signal transduction-related gene expression promoter that is highly safe, has little phytotoxicity, is easy to apply, and exhibits excellent plant disease suppression effects or related gene expression effects that suppress plant disease.

1 is a graph showing the results of gene expression analysis in leaves two days after treatment in evaluation test 3. 1 is a graph showing the results of gene expression analysis in leaves 4 days after treatment in evaluation test 3. In evaluation test 3, FIG. 3(a) is a graph showing the results of gene expression analysis in leaves two days after treatment, and FIG. 3(b) is the results of gene expression analysis in leaves four days after treatment. 1 is a graph showing the results of gene expression analysis in the taproot one day after treatment in evaluation test 4. 1 is a graph showing the results of gene expression analysis in the taproot two days after treatment in evaluation test 4. 1 is a graph showing the results of gene expression analysis in the taproot 4 days after treatment in evaluation test 4. 1 is a graph showing the results of gene expression analysis in the taproot 8 days after treatment in evaluation test 4. FIG. 8 shows the results of gene expression analysis of PR1 in leaves and tap roots. FIG. 9 shows the results of gene expression analysis of P4 in leaves and tap roots.

The following describes an embodiment of the present invention in detail.

(An agent for suppressing plant fungal diseases for use at the soil edge)
The agent for suppressing plant fungal diseases for treatment at the soil edge of the present invention is characterized by containing acetic acid as an active ingredient. The "agent for suppressing plant fungal diseases for treatment at the soil edge of the plant" is an agent for suppressing plant diseases caused by fungi and for treating the soil edge of the plant.

The acetic acid may be the acetic acid contained in vinegar (grain vinegars such as rice vinegar, rice black vinegar, and barley black vinegar, fruit vinegars such as apple vinegar and grape vinegar, synthetic vinegar, distilled vinegar, etc.) or it may be reagent-level acetic acid.

The concentration (acidity) of acetic acid is not particularly limited as long as it is within a range in which a disease suppression effect can be obtained. Although it depends on the type of disease and treatment conditions, for example, the acidity is 0.05% to 2.0%, more preferably 0.05% to 1.0%, 0.1% to 1.0%, or 0.1% to 0.5%.
The acidity (%) is measured according to the "Procedure for measuring the acidity of brewed vinegar" of the Food and Agricultural Materials Inspection and Research Center (FAMIC). In summary, the sample is titrated with 0.5 mol/L sodium hydroxide solution, and the acidity is calculated from the amount of sodium hydroxide solution consumed until the pH reaches 8.2±0.3, converted into acetic acid. Specifically, it is calculated using the following formula (Math. 1).

The plant fungal disease inhibitor for treating the soil edge may contain only acetic acid and water as described above, or may contain components other than acetic acid. For example, it may contain components other than acetic acid contained in vinegar (amino acids, organic acids, alcohols, etc.). Furthermore, it may contain components that are generally blended as plant disease inhibitors (stabilizers, surfactants, pH adjusters, fertilizer components, colorants, flavoring agents, etc.).

The fungal plant diseases of interest are those caused by filamentous fungi. Filamentous fungi are composed of hyphae and are known to inhibit plant growth. Diseases caused by filamentous fungi include powdery mildew, blue mold, red wilt, groove rot, blast, scab, red spot, black spot, gray mold, red burn, yellow patch, yellows, wilt, purple mold, ring spot, gray spot, angular spot, brown rot caused by filamentous fungi, brown spot, brown star, brown spot, brown spot, brown spot, brown spot, brown spot, brown spot, brown spot, stock rot, canker, seedling damping-off, damping-off, downy mildew, half-wilt, late blight, spot disease, black spot, white spot, sesame spot, red spot, sclerotinia disease, black sooty mold, clubroot, white rust, bottom rot, rust, white mold, anthracnose, and root rot.

Plants that are the subject of disease suppression are not particularly limited as long as they are plants that suffer from the above-mentioned diseases. For example, morning glory, aster, astilbe, impatiens, carnation, gerbera, gazania, campanula, bellflower, chrysanthemum, snapdragon, calendula, Christmas rose, clematis, gloxinia, cockscomb, Iceland poppy, cosmos, primula, salvia, zinnia, cineraria, gypsophila, sweet pea, statice, stock, pansy, geranium, delphinium, lisianthus, verbena, sunflower, begonia, Examples include petunias, gentians, lupines, cyclamen, dahlias, tulips, hydrangeas, roses, peonies, kidney beans, tomatoes, cherry tomatoes, eggplants, bell peppers, cucumbers, watermelons, melons, pumpkins, bitter melons, strawberries, Chinese cabbage, cabbage, komatsuna, bok choy, chrysanthemums, okra, shiso, spinach, peas, broad beans, corn, radishes, turnips, carrots, burdock, leeks, onions, asparagus, and potatoes.

The agent for suppressing plant fungal diseases for treating ground-level parts of the present invention is applied to the ground-level parts of plants by spraying, etc. The ground-level part refers to the area at or near the ground, or the boundary between the base of a plant and the ground.
The plant fungal disease inhibitor for treating the ground edge utilizes the fact that, since diseases mainly occur in the stems and leaves, even if the treatment such as spraying is performed only on the ground edge of the plant, the disease inhibitor has a disease inhibitory effect on the stems and leaves. Since the area of the ground edge to be treated by spraying is smaller than that of the stems and leaves, the plant fungal disease inhibitor for treating the ground edge can be efficiently sprayed on the target ground edge as desired. Therefore, the disease inhibitor for treating the ground edge is easy for users to handle. In addition, compared to treatment such as spraying on the stems and leaves, the disease inhibitor after treatment is more likely to remain and remain on the plant (for example, the roots, etc.), and the disease inhibitory effect is more likely to remain after treatment is stopped.

(Method for suppressing plant diseases caused by filamentous fungi)
The method for suppressing a fungal disease in a plant of the present invention includes a step of treating the ground level of a plant with the above-mentioned agent for suppressing a fungal disease in a plant for treating the ground level.

(Plant leaf signal transduction-related gene expression promoter for application at the soil edge)
The plant leaf signaling-related gene expression promoter for use in treatment at the ground edge of the present invention is characterized in that it contains acetic acid as an active ingredient. The "plant leaf signaling-related gene expression promoter for use in treatment at the ground edge of the plant" is an agent that promotes the expression of signaling-related genes in plant leaves and is used for treatment at the ground edge of the plant.

The acetic acid may be the acetic acid contained in vinegar (grain vinegars such as rice vinegar, rice black vinegar, and barley black vinegar, fruit vinegars such as apple vinegar and grape vinegar, synthetic vinegar, distilled vinegar, etc.) or it may be reagent-level acetic acid.

The concentration (acidity) of acetic acid is not particularly limited as long as it is within a range in which the expression of the signal transduction-related gene is promoted. Although it depends on the type of disease, the type of the signal transduction-related gene, and the treatment conditions, the acidity is, for example, 0.05% to 2.0%, more preferably 0.05% to 1.0%, 0.1% to 1.0%, or 0.1% to 0.5%.
The acidity (%) is measured according to the "Procedure for measuring the acidity of brewed vinegar" of the Food and Agricultural Materials Inspection and Research Center (FAMIC). In summary, the sample is titrated with 0.5 mol/L sodium hydroxide solution, and the acidity is calculated from the amount of sodium hydroxide solution consumed until the pH reaches 8.2±0.3, converted into acetic acid. Specifically, it is calculated using the following formula (Math. 1).

The plant leaf signal transduction-related gene expression promoter for use in soil edge treatment may contain only acetic acid and water as described above, or may contain components other than acetic acid. For example, it may contain components other than acetic acid contained in vinegar (amino acids, organic acids, alcohols, etc.). Furthermore, it may contain components that are generally used as plant disease inhibitors (stabilizers, surfactants, pH adjusters, fertilizer components, colorants, flavoring agents, etc.).

The target signaling-related genes are disease resistance-related genes such as salicylic acid signaling-related genes and jasmonic acid signaling-related genes, specifically PR1, PR5, P4 (all salicylic acid signaling-related genes), PR3, PI-II, PR6 (all jasmonic acid signaling-related genes) (see Non-Patent Documents 1 and 2, etc.). Of these, PR1, P4, and PR3 are preferred.

The plant to be subjected to disease suppression is not particularly limited as long as the above-mentioned signal transduction-related genes are promoted in the leaves. For example, tomato is an example.

The plant leaf signaling-related gene expression promoter for use in treating the ground edge of a plant according to the present invention is applied to the ground edge of a plant by spraying, etc. The ground edge refers to the area at or near the ground, or the boundary between the base of a plant and the ground.
The plant leaf signaling-related gene expression promoter for treatment at the ground level has the function of allowing expression of a signaling-related gene, which is a disease-related resistance gene, to be observed at least in the leaves, even when treatment such as spraying is performed only on the ground level of a plant. Since the area of the ground level to be treated by spraying, etc. is smaller than that of stems and leaves, the plant leaf signaling-related gene expression promoter for treatment at the ground level can be efficiently sprayed, etc., as targeted, on the target ground level. For this reason, the plant leaf signaling-related gene expression promoter for treatment at the ground level is easy for users to handle.

(Method for promoting expression of signal transduction-related genes in plant leaves)
The method for promoting leaf signaling-related gene expression in plants of the present invention is characterized by comprising a step of treating the ground edge of a plant with the above-mentioned plant leaf signaling-related gene expression promoter for ground edge treatment.

(Plant main root signal transduction-related gene expression promoter for use at the soil edge)
The promoter for plant main root signaling-related gene expression for use in treating the ground edge of the present invention is characterized in that it contains acetic acid as an active ingredient. The "promoter for plant main root signaling-related gene expression for use in treating the ground edge of the plant" is an agent for promoting the expression of signaling-related genes in the taproot of a plant and for use in treating the ground edge of the plant.

The acetic acid may be the acetic acid contained in vinegar (grain vinegars such as rice vinegar, rice black vinegar, and barley black vinegar, fruit vinegars such as apple vinegar and grape vinegar, synthetic vinegar, distilled vinegar, etc.) or it may be reagent-level acetic acid.

The concentration (acidity) of acetic acid is not particularly limited as long as it is within a range in which the expression of the signal transduction-related gene is promoted. Although it depends on the type of disease, the type of the signal transduction-related gene, and the treatment conditions, the acidity is, for example, 0.05% to 2.0%, more preferably 0.05% to 1.0%, 0.1% to 1.0%, or 0.1% to 0.5%.
The acidity (%) is measured according to the "Procedure for measuring the acidity of brewed vinegar" of the Food and Agricultural Materials Inspection and Research Center (FAMIC). In summary, the sample is titrated with 0.5 mol/L sodium hydroxide solution, and the acidity is calculated from the amount of sodium hydroxide solution consumed until the pH reaches 8.2±0.3, converted into acetic acid. Specifically, it is calculated using the following formula (Math. 1).

The plant main root signal transduction-related gene expression promoter for use in treating the ground edge may contain only acetic acid and water as described above, or may contain components other than acetic acid. For example, it may contain components other than acetic acid contained in vinegar (amino acids, organic acids, alcohols, etc.). Furthermore, it may contain components that are generally formulated as plant disease inhibitors (stabilizers, surfactants, pH adjusters, fertilizer components, colorants, flavoring agents, etc.).

The target signaling-related genes are disease resistance-related genes such as salicylic acid signaling-related genes and jasmonic acid signaling-related genes, specifically PR1, PR5, P4 (all salicylic acid signaling-related genes), PR3, PI-II, PR6 (all jasmonic acid signaling-related genes) (see Non-Patent Documents 1 and 2, etc.). Of these, PR1 or P4 is preferred.

The plant to be subjected to disease suppression is not particularly limited, so long as the above-mentioned signal transduction-related genes are promoted in the taproot. For example, tomato is an example.

The plant main root signaling-related gene expression promoter for use in treating the ground edge of the present invention is applied to the ground edge of a plant by spraying, etc. The ground edge refers to the area at or near the ground, or the boundary between the base of a plant and the ground.
The plant main root signaling-related gene expression promoter for treatment at the ground level has the function of being able to express a signaling-related gene, which is a disease-related resistance gene, at least in the main root, even when treatment such as spraying is performed only at the ground level of a plant. Since the area of the ground level that needs to be treated by spraying, etc. is smaller than that of stems and leaves, the plant main root signaling-related gene expression promoter for treatment at the ground level can be efficiently sprayed, etc., as targeted, to the target ground level. For this reason, the plant main root signaling-related gene expression promoter for treatment at the ground level is easy for users to handle.

(Method for promoting expression of signal transduction-related genes in the primary root of plants)
The method for promoting expression of a main root signaling-related gene in a plant of the present invention is characterized in that it includes a step of treating the ground edge of the plant with the above-mentioned plant main root signaling-related gene expression promoter for ground edge treatment.

(Evaluation Test 1)
The occurrence of powdery mildew on roses was evaluated using an inhibitor of plant fungal diseases for application to the soil edge. A treatment area 1, which was treated at the soil edge, and a non-treated area were defined.

Acetic acid (0.1% acidity aqueous solution) was used as the disease inhibitor. Roses (variety: Mallorca, No. 6 long pot, 2-year-old seedling, height 50 cm) were planted in three plots, and the treatment was repeated three times.

Disease-free roses were treated with disease inhibitors using the treatment method for each test plot. Since no disease developed, one week after the start of treatment, they were inoculated using the dusting method using leaves infected with powdery mildew.

The treatment at the base of the plant was carried out using a spray trigger at the base of the plant 30 times per plant (30 mL).
Table 1 shows each treatment area and its treatment overview.

After the start of the treatment, the treatment was carried out at intervals of once every 2-3 days, and the phytotoxicity test was carried out six times and the efficacy test was carried out four times.
The treatment date, inoculation date, drug damage investigation date, and drug efficacy investigation date are shown in Table 2.

The damage caused by the drug was examined by visual inspection.
Each survey day was evaluated according to the "Pesticide Damage Survey Standards" below. The Pesticide Damage Survey Standards are based on the New Pesticide Practical Tests published by the Japan Plant Protection Association. In addition, a comprehensive evaluation was made according to the "Pesticide Damage Evaluation Standards" below.

"Drug Damage Investigation Standards"
-: No drug-related harm is acknowledged.
+: Minor drug-related symptoms observed.
++: Moderate drug-related symptoms observed.
+++: Severe drug-related symptoms observed.

"Evaluation Criteria for Drug-Related Injuries"
-: No adverse effects.
±: Drug damage was observed, but not to the extent that would cause problems in practical use.
+: Damage caused by the drug is evident and is problematic for practical use.
Symptoms of phytotoxicity are judged by discoloration or withering of leaves or petals, shrinkage or withering of the growing point, withering or death of the entire plant, etc. Whether or not there is a problem in practical use is judged by whether or not there is an effect on the growth of the plant.

The results of the drug damage investigation are shown in Table 3.

As shown in Table 3, in treatment area 1, where only the soil edge was treated, no phytotoxicity was observed, just like in the untreated area, and it was evaluated as having no phytotoxicity.

Next, to evaluate efficacy, approximately 100 random leaflets per plant were selected to investigate the number of diseased leaves by severity, and the diseased leaf rate (%) was calculated using the formula below (Number 2), and the disease severity (5) was calculated using the formula below (Number 3).

"Number of diseased leaves by severity"
Index 0: No disease is observed.
Index 1: Lesion area occupies less than 5% of the leaf area.
Index 2: The lesion area occupies 5% or more but less than 25% of the leaf area.
Index 3: The lesion area occupies 25% or more but less than 50% of the leaf area.
Index 4: The lesion area occupies 50% or more of the leaf area.

The control value was calculated from the calculated disease incidence (%) using the following formula (Number 4).

The survey results for the number of diseased leaves by severity, and the calculated diseased leaf rate, disease severity, and control value are shown in Tables 5 to 7. Table 5 shows the results 22 days after the start of treatment (3 days after 9 treatments), Table 6 shows the results 28 days after the start of treatment (6 days after 10 treatments), and Table 7 shows the results 35 days after the start of treatment (13 days after 10 treatments).
Table 4 shows the control value and the disease severity without treatment.

From Tables 3 to 7, in treatment area 1 (treatment at the ground level), the control value was 51.7 6 days after the 10th treatment (28 days after treatment started), and even 13 days after the 10th treatment (35 days after treatment started), the control value remained at 38.7. These results show that treatment at the ground level also has a disease suppressing effect against powdery mildew, a fungal disease that causes damage to leaves.

(Evaluation Test 2)
The incidence of powdery mildew on cucumber was evaluated using an inhibitor for fungal diseases caused by plant root zone treatment. A treatment zone 1, which was treated at the root zone, and a non-treated zone were defined.

Acetic acid (0.1% acidity aqueous solution) was used as the disease inhibitor. Cucumbers (variety: scion Nina (S-27), rootstock: GT-2, growth stage: 4-5 leaf stage) were planted in 4 plants per plot, and the treatment was repeated 3 times.

Cucumber seedlings that were free of powdery mildew were treated as described below and then planted in No. 6 pots. After planting, fertilization and watering were carried out in the same manner as conventional cultivation, and each treatment method was continued. After the onset of powdery mildew, surveys were conducted four times.

The treatment at the base of the plant was carried out using a spray trigger at the base of the plant 30 times per plant (30 mL).
Table 8 shows each treatment area and its outline.

After the start of the treatment, the treatment was carried out at intervals of once every 2-3 days, and the phytotoxicity and efficacy were examined four times each.
The treatment date, inoculation date, drug damage investigation date, and drug efficacy investigation date are shown in Table 9.

The damage caused by the drug was examined by visual inspection.
Each day of the survey, the results were evaluated according to the "Chemical Damage Survey Criteria" below. In addition, the results were comprehensively evaluated according to the "Chemical Damage Evaluation Criteria" below.

"Drug Damage Investigation Standards"
-: No drug-related harm is acknowledged.
+: Minor drug-related symptoms observed.
++: Moderate drug-related symptoms observed.
+++: Severe drug-related symptoms observed.

"Evaluation Criteria for Drug-Related Injuries"
-: No adverse effects.
±: Drug damage was observed, but not to the extent that would cause problems in practical use.
+: Damage caused by the drug is evident and is problematic for practical use.
Symptoms of phytotoxicity are judged by discoloration or withering of leaves or petals, shrinkage or withering of the growing point, withering or death of the entire plant, etc. Whether or not there is a problem in practical use is judged by whether or not there is an effect on the growth of the plant.

The results of the drug damage investigation are shown in Table 10.

As shown in Table 10, in treatment area 1, where only the soil edge was treated, no phytotoxicity was observed, just like in the untreated area, and it was evaluated as having no phytotoxicity.

Next, regarding efficacy, the number of diseased leaves by severity was investigated for approximately 34 to 92 leaves per plant, and the diseased leaf rate (%) was calculated using Equation 2, and the disease severity (%) was calculated using Equation 3.

"Number of diseased leaves by severity"
Index 0: No disease is observed.
Index 1: Lesion area occupies less than 5% of the leaf area.
Index 2: The lesion area occupies 5% or more but less than 25% of the leaf area.
Index 3: The lesion area occupies 25% or more but less than 50% of the leaf area.
Index 4: The lesion area occupies 50% or more of the leaf area.

The control value was calculated from the calculated disease incidence (%) using equation 4.

The survey results for the number of diseased leaves by severity, and the calculated diseased leaf rate, disease severity, and control value are shown in Tables 11 to 14. Table 12 shows the results 8 days after the start of treatment, Table 13 shows the results 14 days after the start of treatment, and Table 14 shows the results 18 days after the start of treatment.
Table 11 shows the control values.

As can be seen from Tables 11 to 14, in treatment area 1 (treatment at the ground level), the control value was 20.1 8 days after the start of treatment, 16.6 14 days after the start of treatment, and 10.5 18 days after the start of treatment. These results confirmed that treatment at the ground level also had a higher disease suppression effect than the untreated area against powdery mildew, a fungal disease that causes damage on the leaves, and demonstrated that treatment at the ground level can suppress disease that occurs on the leaves.

(Evaluation Test 3)
We analyzed the expression of tomato signal transduction-related genes, which are related to disease resistance, using a plant leaf signal transduction-related gene expression promoter for application at the soil edge.

Tomato seeds (variety: Home Momotaro) were sown in a 128-well cell tray filled with sterilized horticultural soil, and then grown for 4 weeks in a greenhouse (conditions: watering with distilled water as needed, irrigation with liquid fertilizer after 2 weeks).
Each treatment shown in Table 15 was applied to the soil edge of each pot.
In treatment area 1, about 2.4 ml of acetic acid (aqueous solution with an acidity of 0.1%) was sprayed onto the roots of the tomatoes per cell (volume 24 ml).
In treatment area 2, ASM (acibenzolar-S-methyl) (concentration 20 ppm) containing 0.01% by volume of Tween 20 (polyoxyethylene sorbitan monolaurate) polyoxyethylene sorbitan monolaurate was sprayed. Approximately 2.4 ml was sprayed per treatment area.
In the untreated area, about 2.4 ml of water was sprayed.
The processing volume of 2.4 ml per cell corresponds to 30 sprays (30 mL) per 9 cm pot (300 ml capacity).

After 1, 2, 4, and 8 days, the primary leaves (0.07g-0.1g) were immediately placed in liquid nitrogen and stored at -80°C.

RNA was extracted using Isogen (manufactured by Nippon Gene Co., Ltd.) according to the protocol. DNA in the RNA was degraded by Dnase I treatment using RQ1 (manufactured by Promega Co., Ltd.). Complementary DNA (cDNA) was synthesized using PrimeScript RT reagent kit (manufactured by Takara Co., Ltd.).
Using SYBR Premix Ex Taq II manufactured by Takara Biosciences, quantitative analysis was performed by real-time PCR targeting the target genes and the actin gene, which is a reference gene, related to disease resistance in tomato, as shown below, and the relative expression level of each target gene was calculated by correcting it with the expression level of the reference gene.

The target genes are as follows:
"Salicylic acid signaling-related genes"
PR1
PR5
P4
"Jasmonic acid signaling-related genes"
PR3
P-II
PR6

PCR (polymerase chain reaction) was performed using a StepOnePlus kit manufactured by Applied Biosystems under the following reaction conditions: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds (40 cycles), 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds.

Gene expression analysis was performed in the leaves 2 days and 4 days after treatment. The results after 2 days are shown in Table 16 and Figure 1, and the results after 4 days are shown in Table 17 and Figure 2. The gene expression levels were expressed as relative values with the untreated area set as 1.

From Table 16 and Figure 1, the increase in gene expression level two days after treatment in treatment areas 1 and 2 was at most six times that of the untreated area.

From Table 17 and Figure 2, the increase in gene expression levels in treatment areas 1 and 2 4 days after treatment was greater than that after 2 days for all target genes, with the expression levels of the P4 gene in treatment area 1 being approximately 48 times that of the untreated area, the PR3 gene being approximately 24 times that of the untreated area, and the PR1 gene being approximately 10 times that of the untreated area.

To clarify the comparison between treated area 1 and the untreated area, we performed a comparison of expression levels and a statistical analysis (Wilcoxon rank test) between treated area 1 and the untreated area. As shown in Figure 3, it was confirmed that the expression levels of PR1, P4, and PR3 genes increased significantly at p<0.05 4 days after treatment.

(Evaluation Test 4)
Using a plant main root signal transduction-related gene expression promoter for application at the soil edge, we analyzed the expression of tomato signal transduction-related genes, which are related to disease resistance.

Tomato seeds (variety: Home Momotaro) were sown in a 128-hole cell tray filled with sterilized horticultural soil (Nippi Pp horticultural soil) and then grown for 4 weeks in a greenhouse (conditions: watering with distilled water as needed, irrigation with a 1000-fold solution of liquid fertilizer Hyponex after 2 weeks).
Each treatment shown in Table 18 was applied to the soil edge of each pot.
In treatment area 1, about 2.4 ml of acetic acid (aqueous solution with an acidity of 0.1%) was sprayed onto the roots of the tomatoes per cell (volume 24 ml).
In treatment area 2, ASM (acibenzolar-S-methyl) (concentration 20 ppm) containing 0.01% by volume of Tween 20 (polyoxyethylene sorbitan monolaurate) polyoxyethylene sorbitan monolaurate was sprayed. Approximately 2.4 ml was sprayed per treatment area.
In the untreated area, about 2.4 ml of water was sprayed.
The processing volume of 2.4 ml per cell corresponds to 30 sprays (30 mL) per 9 cm pot (300 ml capacity).

After 1, 2, 4, and 8 days, the main root (which was wiped dry with a paper cloth such as Kimwipe and then cut 5 to 7 mm from the base) was immediately placed in liquid nitrogen and stored at minus 80 degrees Celsius.

RNA was extracted using Isogen (manufactured by Nippon Gene Co., Ltd.) according to the protocol. DNA in the RNA was degraded by Dnase I treatment using RQ1 (manufactured by Promega Co., Ltd.). Complementary DNA (cDNA) was synthesized using PrimeScript RT reagent kit (manufactured by Takara Co., Ltd.).
Using SYBR Premix Ex Taq II manufactured by Takara Biosciences, quantitative analysis was performed by real-time PCR targeting the target genes and the actin gene, which is a reference gene, related to disease resistance in tomato, as shown below, and the relative expression level of each target gene was calculated by correcting it with the expression level of the reference gene.

The target genes are as follows:
"Salicylic acid signaling-related genes"
PR1
PR5
P4
"Jasmonic acid signaling-related genes"
PR3
P-II
PR6

PCR (polymerase chain reaction) was performed using a StepOnePlus kit manufactured by Applied Biosystems under the following reaction conditions: 95°C for 30 seconds, 95°C for 5 seconds, 60°C for 30 seconds (40 cycles), 95°C for 15 seconds, 60°C for 60 seconds, and 95°C for 15 seconds.

Gene expression analysis was performed on the main root 1, 2, 4, and 8 days after treatment. The results after 1 day are shown in Table 19 and Figure 4, the results after 2 days are shown in Table 20 and Figure 5, the results after 4 days are shown in Table 21 and Figure 6, and the results after 8 days are shown in Table 22 and Figure 7. Gene expression levels were expressed as relative values with the untreated group set to 1.

As can be seen from Table 19 and Figure 4, an increase in gene expression level was observed for all target genes one day after treatment in treatment areas 1 and 2. In particular, a significant increase of approximately 28 times was observed for the PR1 gene in treatment area 1 compared to the untreated area, and approximately 25 times was observed for the P4 gene.

From Table 20 and Figure 5, a tendency for gene expression levels to increase two days after treatment in treatment areas 1 and 2 was observed for all target genes. In particular, the PR1 gene in treatment area 1 showed a significant increase of about 12 times that of the untreated area, and the P4 gene showed a significant increase of about 19 times that of the untreated area, but this was smaller than the increase in gene expression levels one day after treatment.

From Table 21 and Figure 6, the increase in gene expression level 4 days after treatment in treatment area 1 was generally small, with the PR1 gene increasing by about 4 times compared to the untreated area, the P4 gene increasing by about 5 times compared to the untreated area, and the PR6 gene increasing by about 3 times compared to the untreated area, with no significant difference from the untreated area for other genes. The increase in gene expression level in treatment area 2 (positive control) 4 days after treatment was approximately 24 times compared to the untreated area for the PR1 gene, approximately 19 times compared to the untreated area for the P4 gene, and approximately 3 times compared to the untreated area for the PR6 gene, with no significant difference from the untreated area for other genes.

From Table 22 and Figure 7, the increase in gene expression levels 8 days after treatment in treatment areas 1 and 2 was generally small, with the maximum increase being about 2 to 3 times that of the untreated area.

(Summary of Evaluation Test 3 and Evaluation Test 4)
From the above evaluation tests 3 and 4, it was confirmed that when the ground part of a plant was treated (sprayed at the ground part) with a signal transduction-related gene expression promoter containing acetic acid as an active ingredient, the expression levels of PR1 gene and P4 gene increased in both leaves and taproot. The time course of the expression level of PR1 gene is shown in Table 23 and Figure 8, and the time course of the expression level of P4 gene is shown in Table 24 and Figure 9.

From Tables 23 and 24 and Figures 8 and 9, it was found that in the leaves, an increase in gene expression was observed from 2 days to 4 days after treatment, with the gene expression level tending to be maximum 4 days after treatment. On the other hand, in the main root, the gene expression level was maximum 1 day after treatment, and thereafter tended to decrease over time. In other words, it was found that these gene expressions first occurred promptly in the main root, and then occurred later in the leaves.

The present invention may have the following configurations.
[Item 1]
An agent for suppressing plant fungal diseases for application at the soil edge, which contains acetic acid as an active ingredient.
[Item 2]
2. The agent for suppressing a plant fungal disease for use in treating soil edge according to item 1, wherein the fungal disease is powdery mildew.
[Item 3]
3. The agent for suppressing a disease caused by a filamentous fungus for application to a soil edge of a plant according to item 1 or 2, wherein the plant is a rose or a cucumber.
[Item 4]
4. The agent for suppressing plant fungal diseases for application to soil edge according to any one of items 1 to 3, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less.
[Item 5]
5. A method for suppressing a fungal disease in a plant, comprising a step of treating a ground edge of the plant with the agent for suppressing a fungal disease in a plant according to any one of items 1 to 4.
[Item 6]
A plant leaf signal transduction-related gene expression promoter for application at the soil edge, containing acetic acid as an active ingredient.
[Item 7]
7. The plant leaf signaling-related gene expression promoter for use in application at the ground edge according to Item 6, wherein the signaling-related gene is PR1, P4 or PR3.
[Item 8]
8. The plant leaf signal transduction-related gene expression promoter for application to ground edge according to item 6 or 7, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less.
[Item 9]
Item 9. The plant leaf signal transduction-related gene expression promoter for use in treating ground edge according to any one of Items 6 to 8, wherein the plant is a tomato.
[Item 10]
10. A method for promoting expression of a leaf signaling-related gene in a plant, comprising a step of treating a ground edge of the plant with the plant leaf signaling-related gene expression promoter for treatment of the ground edge according to any one of items 6 to 9.
[Item 11]
A promoter for signal transduction-related gene expression in the main root of plants for application at the soil edge, containing acetic acid as an active ingredient.
[Item 12]
Item 12. The plant main root signaling-related gene expression promoter for use in treating the ground edge according to Item 11, wherein the signaling-related gene is PR1 or P4.
[Item 13]
Item 13. The plant main root signaling-related gene expression promoter for use in treating a ground edge according to item 11 or 12, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less.
[Item 14]
Item 14. The plant main root signaling-related gene expression promoter for use in treating the ground edge according to any one of Items 11 to 13, wherein the plant is a tomato.
[Item 15]
Item 15. A method for promoting expression of a main root signaling-related gene in a plant, comprising treating a ground edge of the plant with the plant main root signaling-related gene expression promoter for treatment of the ground edge according to any one of Items 11 to 14.

Claims (15)

An inhibitor of fungal plant diseases for use at the soil edge, containing acetic acid as the active ingredient. The fungal disease inhibitor for treating soil edge according to claim 1, wherein the fungal disease is powdery mildew. The plant fungal disease inhibitor for soil edge treatment according to claim 1 or 2, wherein the plant is a rose or a cucumber. The fungal disease inhibitor for use in treating soil edge according to claim 1 or 2, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less. A method for suppressing a fungal disease in a plant, comprising a step of treating the soil edge of the plant with the agent for suppressing a fungal disease caused by a fungus for treating the soil edge of the plant according to claim 1 or 2. A promoter for gene expression related to signal transduction in plant leaves for application at the soil edge, containing acetic acid as the active ingredient. The plant leaf signaling-related gene expression promoter for use in soil edge treatment according to claim 6, wherein the signaling-related gene is PR1, P4 or PR3. The plant leaf signal transduction-related gene expression promoter for use in soil edge treatment according to claim 6 or 7, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less. The plant leaf signal transduction-related gene expression promoter for use in treating the soil edge according to claim 6 or 7, wherein the plant is a tomato. A method for promoting expression of a leaf signaling-related gene in a plant, comprising a step of treating the ground edge of the plant with the plant leaf signaling-related gene expression promoter for treatment of the ground edge according to claim 6. A promoter for signal transduction-related gene expression in the main root of plants for application at the soil edge, containing acetic acid as the active ingredient. The plant main root signaling-related gene expression promoter for use in treating the ground edge according to claim 11, wherein the signaling-related gene is PR1 or P4. The plant main root signal transduction-related gene expression promoter for use in treating the ground edge according to claim 11 or 12, wherein the acidity of the acetic acid is 0.05% or more and 2.0% or less. The plant main root signal transduction-related gene expression promoter for use in treating the ground edge according to claim 11 or 12, wherein the plant is a tomato. A method for promoting the expression of a plant's main root signaling-related gene, comprising a step of treating the plant's ground edge with the plant main root signaling-related gene expression promoter for treating the ground edge according to claim 11.

Images