Composition for preventing the onset of clubroot disease

Abstract

We propose a novel method for controlling clubroot disease. [Solution] This invention provides a composition containing wood vinegar for preventing the onset of clubroot disease in plants.

Description

Technical Field

[0001] The present invention relates to a composition for preventing the onset of clubroot disease, a method for preventing the onset of clubroot disease, a composition for improving plant cultivation soil contaminated with clubroot pathogen, and a method for improving plant cultivation soil contaminated with clubroot pathogen.

Background Art

[0002] Clubroot pathogen (Plsamodiophora brassicae) is a soil-borne pathogen belonging to the phylum Plasmodiophoromycota and is an obligate parasitic protozoan. This bacterium mainly infects cruciferous plants and forms knots of various sizes on the roots. Plants affected by clubroot pathogen have reduced ability to absorb water and nutrients, resulting in wilting of the above-ground part of the plant, growth inhibition, and sometimes death. Clubroot pathogen lives as dormant spores in the soil. When the roots of cruciferous plants approach these dormant spores, the dormant spores germinate into primary zoospores due to some stimulus. When this infects the root hair, a zoosporangium of the zoospore is formed in the cells of the root hair, and secondary zoospores are formed therein. When these secondary zoospores are released from the root hair and infect the cortical cells of the main root or lateral root, they proliferate there and cause abnormal proliferation of the root cells. As a result, the supply of water and nutrients from the soil to the above-ground part is inhibited because the vascular bundle is compressed, so the growth of the above-ground part of the plant is significantly suppressed.

[0003] As general measures against clubroot disease, improvement of the soil environment by incorporating lime materials such as calcium cyanamide, magnesium lime, and oyster shells into the soil, and use of agricultural chemicals (fungicides) such as flusulfamide, fluazinam, amisulbrom, and cyazofamid are carried out (for example, Patent Document 1).

[0004]

Prior Art Documents

Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] While lime-based soil pH correction can mitigate some soil-borne diseases, its effectiveness is limited to cases where the disease is mild. Furthermore, the use of pesticides can kill even beneficial, non-pathogenic microorganisms, and can lead to pesticide residue problems in the soil. [Means for solving the problem]

[0007] The inventors of this invention investigated novel methods for controlling clubroot disease and found that the use of wood vinegar is effective.

[0008] In other words, the present invention relates in one embodiment to a composition for preventing the onset of clubroot disease in plants, comprising wood vinegar.

[0009] Another embodiment of the present invention relates to a composition for improving soil for plant cultivation that is contaminated with clubroot fungus, comprising wood vinegar.

[0010] In one embodiment of the present invention, the plant is characterized in that it is a plant of the Brassicaceae family.

[0011] In one embodiment of the present invention, the Brassicaceae plant is characterized in that it is a Brassicaceae vegetable.

[0012] In one embodiment of the present invention, the cruciferous vegetable is a vegetable selected from the group consisting of Chinese cabbage, cabbage, Brussels sprouts, mizuna, komatsuna, turnip, broccoli, cauliflower, bok choy, nozawana, tsukena, nabana, tatsoi, rapeseed, mustard greens, takana, watercress, radish, and kale.

[0013] In one embodiment of the present invention, the composition is applied to soil for plant cultivation such that the concentration of wood vinegar in the soil is 0.05 to 5.0 w / w%.

[0014] In one embodiment of the present invention, it is characterized in that the soil does not contain organic fertilizer.

[0015] Another embodiment of the present invention relates to a method for preventing the onset of root knot disease in plants, which includes the step of applying wood vinegar to the soil in which the plants are cultivated or the soil to be cultivated with the plants.

[0016] Another embodiment of the present invention relates to a method for improving soil contaminated with root knot disease bacteria, which includes the step of applying wood vinegar to the soil contaminated with the root knot disease bacteria.

[0017] Inventions that arbitrarily combine one or more of the features of the present invention listed above are also included in the scope of the present invention.

Brief Description of the Drawings

[0018] [Figure 1] Figure 1 is a microscopic photograph of root knot disease bacteria dormant spores. [Figure 2] Figure 2 shows the cut part of the plant body for the growth analysis of Komatsuna. [Figure 3] Figure 3 shows the classification of the incidence degree of root knot disease of Komatsuna. [Figure 4] Figure 4 shows the transition of the number of soil bacteria in organic soil or chemical soil. [Figure 5] Figure 5 shows the growth state of Komatsuna in each soil. [Figure 6] Figure 6 shows the growth state of Komatsuna roots in diseased soil. [Figure 7] Figure 7 shows the incidence degree of root knot disease in diseased soil of chemical soil and organic soil. [Figure 8] Figure 8 shows the effect of wood vinegar in chemical soil. [Figure 9] Figure 9 shows the effect of wood vinegar in organic soil. [Figure 10] Figure 10 shows the effect of wood vinegar in chemical soil. [Figure 11] Figure 11 shows the effect of wood vinegar in organic soil. [Figure 12] Figure 12 shows the changes in the soil bacterial flora caused by wood vinegar. [Modes for carrying out the invention]

[0019] This invention is based on novel findings that the use of wood vinegar can prevent the onset of clubroot disease in plants, or improve soil contaminated with clubroot fungi.

[0020] Wood vinegar may be crude wood vinegar obtained by dry distillation (carbonization) and standing of woody raw materials, or it may be obtained by removing light oils and tar components from crude wood vinegar by standing or distillation. Alternatively, wood vinegar may be obtained by mixing multiple crude raw materials of different concentrations, obtained by distilling crude wood vinegar obtained by dry distillation and standing of woody raw materials, to adjust the concentration and components. Examples of woody raw materials include trees, bamboo, palm, straw, and rice husks. Wood vinegar may contain various organic components, including organic acids such as acetic acid and phenols (e.g., guaiacol, phenol, 4-methylguaiacol). The proportion of components contained in wood vinegar may vary to some extent depending on the raw materials and the number of distillation cycles, but any wood vinegar that is sold as "wood vinegar" on the market and is generally available can be used in this invention.

[0021] The following describes a non-restrictive method for producing wood vinegar. When wood gas generated by dry distillation of woody raw materials is cooled, a liquid substance is produced. When this liquid substance is allowed to stand, it separates, and crude wood vinegar is obtained from the upper layer and wood tar from the lower layer. The crude wood vinegar is distilled to remove harmful substances and prepare several crude raw materials of different concentrations. Crude raw materials of varying concentrations are obtained by distillation. Distillation may be repeated several times (preferably two or more times) to prepare a concentrated grade of crude raw material.

[0022] The aforementioned crude materials are mixed and blended to prepare wood vinegar with a desired acetic acid concentration (e.g., about 4-10%). The concentration of the wood vinegar can also be evaluated based on the concentrations of guaiacol, phenol, 4-methylguaiacol, etc., in addition to the acetic acid contained. The concentration of each component may be measured, for example, using gas chromatography.

[0023] A more specific example of a method for producing wood vinegar is described below. When woody raw materials are dry-distilled and the resulting wood gas is cooled, a liquid substance is produced. This liquid substance is subjected to vacuum distillation at a liquid temperature of 100°C or less in a first vessel (for example, a stainless steel distillation vessel). By allowing this crude distillate to stand for several days (preferably 10 days or more), it is separated into three layers: a light oil layer, an aqueous layer, and a settled tar layer. Only the aqueous layer, which is mainly composed of acetic acid, is extracted as crude wood vinegar.

[0024] Next, the crude wood vinegar (aqueous layer) is subjected to atmospheric distillation in a second pot at a liquid temperature of approximately 100°C. In this operation, the crude wood vinegar is placed in a distillation pot to remove the initial distillate. This distillation removes low-boiling-point substances such as methanol, acetone, and aldehydes, and also removes harmful substances (such as benzo(a)pyrene) that may form azeotropes with these low-boiling-point substances.

[0025] Once the initial distillate of the crude wood vinegar placed in the second distillation still has been removed and about half of the remaining amount has been distilled, the distillation is stopped and the remaining half of the crude wood vinegar in the still is transferred to the third still. The remaining half of the distilled amount may be returned to the second still and distilled again. This process yields a crude raw material from which low-boiling point substances and benzo(a)pyrene and other substances that may form azeotropes with them have been more carefully and reliably removed from the second still.

[0026] After removing low-boiling-point substances, approximately half of the remaining liquid in the second pot is distilled at atmospheric pressure in the third pot at a liquid temperature of about 100-120°C to efficiently recover the fraction containing acetic acid, the main component of wood vinegar, thereby obtaining high-purity distilled and refined wood vinegar as the crude raw material.

[0027] In this way, multiple crude raw materials with varying concentrations are prepared. These multiple crude raw materials are then mixed and blended to achieve the desired component concentration (for example, an acetic acid concentration of approximately 4-10%) to obtain the desired wood vinegar.

[0028] In short, the wood vinegar that may be used in the present invention may be produced by the following method: A liquid substance obtained by dry distillation of woody raw materials is subjected to vacuum distillation, and the crude distillate obtained by vacuum distillation is allowed to stand to separate it into three layers: a light oil layer, an aqueous layer, and a settled tar layer. The aqueous layer is removed, and the removed crude wood vinegar is subjected to at least two atmospheric distillations to recover the fraction containing acetic acid, which is the main component of wood vinegar, resulting in wood vinegar with an acetic acid concentration of 4-10%.

[0029] Clubroot disease is caused by infection with Plsamodiophora brassicae, a soilborne pathogen belonging to the phylum Plsamodiophora. Clubroot disease is a plant disease that mainly affects Brassicaceae plants. Plants infected with clubroot develop small and large galls on their roots, reducing their ability to absorb nutrients and water, leading to wilting, stunted growth, and sometimes death of the above-ground parts of the plant.

[0030] The plants targeted by this invention may be cruciferous vegetables, particularly those of the Brassicaceae family that have high value as edible vegetables. Examples of cruciferous vegetables include Chinese cabbage, cabbage, Brussels sprouts, mizuna, komatsuna, turnip, broccoli, cauliflower, bok choy, nozawana, tsukena, nabana, tatsoi, rapeseed, mustard greens, takana, watercress, daikon radish, and kale.

[0031] The composition or method of the present invention can be used in soil containing organic fertilizers (organic soil) and soil without organic fertilizers (chemical soil). However, soil without organic fertilizers is soil in which there are almost no soil bacteria that can suppress the growth of clubroot fungi, and if no countermeasures are taken, clubroot disease is likely to spread, so the effects of the present invention may be more pronounced. It is thought that by applying wood vinegar, for example, changes in the soil microbial flora may occur, and the desired effect may be obtained.

[0032] In the present invention, the concentration of wood vinegar applied to the soil may be appropriately adjusted by those skilled in the art depending on the type of plant and the degree of disease caused by clubroot fungus, but for example, it may be 0.05 to 5.0 w / w% (concentration of wood vinegar relative to the soil). The lower limit of the wood vinegar concentration may be, for example, 0.05 w / w%, 0.1 w / w%, 0.2 w / w%, 0.3 w / w%, 0.4 w / w%, or 0.5 w / w%, and the upper limit of the wood vinegar concentration may be, for example, 5.0 w / w%, 4.5 w / w%, 4.0 w / w%, 3.5 w / w%, 3.0 w / w%, 2.5 w / w%, 2.0 w / w%, 1.5 w / w%, or 1.0 w / w%. If the concentration of wood vinegar in the soil falls below 0.05 w / w%, the effects of the present invention may not be fully obtained. If the concentration of wood vinegar in the soil exceeds 5.0 w / w%, adverse effects on plants, soil, and beneficial microorganisms in the soil may occur.

[0033] As for the application of wood vinegar, it may be mixed or blended into the soil, or drenched or sprayed onto the soil. Alternatively, wood vinegar or its diluted solution may be adsorbed onto a porous material (e.g., charcoal, bamboo charcoal, zeolite, sepiolite, silica gel, bentonite, diatomaceous earth, etc.) before use. Or, wood vinegar may be dried and processed into a powder or granules before use.

[0034] The present invention can be used as wood vinegar alone, but it may also be used in combination with other compositions and methods useful for suppressing clubroot disease. For example, in addition to the present invention, soil environment improvement may be carried out by incorporating lime materials such as calcium cyanamide, magnesium lime, and oyster shells into the soil, or mineral-containing materials, compost, amino acid-containing materials, peptide-containing materials, and protein-containing materials may be applied. Furthermore, beneficial microorganisms may be applied to the soil. Examples of beneficial microorganisms include Agrobacterium, Erwinia, Enterobacter, Xanthomonas, Gliocladium, Coniothyrium, Pseudomonas, Streptomyces, and Talaromyces. Examples of microorganisms include those belonging to the genera *Omyces*, *Trichoderma*, *Burkholderia*, *Bacillus*, *Variovorax*, *Pythium*, *Fusarium*, *Penicillium*, *Lactobacillus*, *Rhizoctonia*, and mycorrhizal fungi. In particular, Agrobacterium radiobacter, Bacillus subtilis, Bacillus thuringiensis, Burkholderia cepacia, Coniothyrium minitans, Enterobacter cloacae, and non-pathogenic Erwinia carotovora subsp.carotovora), Erwinia herbicola, Fusarium equiseti, Fusarium oxysporum, Fusarium solani, Lactobacillus kyotoensis, Lactobacillus plantarum, Penicillium oxalicum, Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pythium oligandrum, Pythium acanthofolon Examples of microorganisms belonging to the following groups include *acanthophoron*, *Pythium mycoparasiticum*, *Binucleate Rhizoctonia sp.*, *Streptomyces galbus*, *Talaromyces flavus*, *Tricoderma atroviride*, *Trichoderma harzianum*, *Trichoderma viride*, *Variovorax paradoxus*, *Xanthomonas campestris*, and *Xanthomonas maltophilia*.

[0035] The terms used herein are for the purpose of describing specific embodiments and are not intended to limit the invention.

[0036] Furthermore, the term "includes" as used herein, unless the context clearly indicates otherwise, means that the described items (components, steps, elements, or numbers, etc.) are present, and does not exclude the presence of other items (components, steps, elements, or numbers, etc.).

[0037] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as they are commonly understood by those skilled in the art to which this invention pertains. Unless otherwise explicitly defined, terms used herein should be interpreted as having meaning consistent with their meaning in this specification and in the related art, and should not be interpreted in an idealized or overly formal sense.

[0038] Embodiments of the present invention may be described with reference to schematic diagrams, but in the case of schematic diagrams, they may be exaggerated in order to clarify the explanation.

[0039] In this specification, for example, when expressed as "1 to 10 w / w%", a person skilled in the art will understand that such expression refers specifically to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 w / w%.

[0040] In this specification, any numerical values ​​used to indicate ingredient content or numerical ranges shall be interpreted as including the meaning of the term "approximately" unless otherwise specified. For example, "10 times" shall be understood to mean "approximately 10 times" unless otherwise specified.

[0041] All disclosures of the documents cited herein should be deemed to be incorporated herein by reference, and those skilled in the art will understand, in accordance with the context herein, the relevant disclosures in those prior art documents as part of this specification without departing from the spirit and scope of the invention. [Examples]

[0042] 1. Experimental materials and methods

[0043] (1) Preparation of base soil Vermiculite (Togawa Heiwa Farm, Tochigi), woody soil (Daiken Corporation, Osaka), mountain soil (Ishihara, Aichi), peat moss (Togawa Heiwa Farm, Tochigi), and black soil (Togawa Heiwa Farm, Tochigi) were dried and mixed in the volume ratios shown in Table 1 to form the base soil. [Table 1]

[0044] (2) Soil preparation using organic materials Cow manure compost (Taniguchi Farm, Shiga), soybean meal (processed soybeans, J-Oil Mills, Tokyo), oilseed meal (rapeseed oilseed meal, JOY Agris, Tokyo), and bone meal (Higashiyama Bussan, Osaka) were dried and finely crushed. These organic materials were thoroughly mixed with the base soil in the mass ratios shown in Table 2 relative to the weight of the base soil to create "organic soil." [Table 2]

[0045] (3) Soil preparation using chemical fertilizers A "chemical soil" was created by applying powdered ammonium sulfate, superphosphate, and potassium sulfate (all from Kimura Green, Shiga) to the base soil at concentrations of N: 100 mg / kg, P: 100 mg / kg, and K: 100 mg / kg.

[0046] (4) Analysis of the total number of bacteria in the soil 1 g of soil sample was weighed into a 50 mL centrifuge tube that had been autoclaved (121°C, 20 min), and 8.0 mL of DNA extraction buffer (Table 3) and 1.0 mL of 20% sodium dodecyl sulfate (SDS) solution were added. The centrifuge tube was placed in a stirrer and stirred (1,500 rpm, 20 min, room temperature). 1.5 mL of the sample mixture was transferred from the centrifuge tube into a 2.0 mL tube and centrifuged using a centrifuge (KUBOTA 3700, Kubota, Tokyo) (8,000 rpm, 20°C, 10 min). 700 μL of the aqueous layer was transferred into a 1.5 mL organic solvent-resistant tube, 700 μL of chloroform-isoamyl alcohol (24:1, v / v) was added, and after gentle stirring, the mixture was centrifuged (14,000 rpm, 20°C, 10 min). 500 μL of the aqueous layer was transferred to a 1.5 mL tube, 300 μL of 2-propanol was added, and the mixture was gently stirred before centrifugation (14,000 rpm, 20°C, 20 min). The aqueous layer was removed, 1.0 mL of 70% (v / v) ethanol was added, and the mixture was centrifugated (14,000 rpm, 20°C, 5 min). The aqueous layer was removed, and the mixture was dried under reduced pressure for 30 minutes using an aspirator (AS-01, ASONE, Osaka). 50 μL of 10:1 TE buffer (Table 4) was added to suspend the mixture, and this was used as the eDNA extract.

[0047] Next, the total bacterial count of the eDNA extract was quantified by electrophoresis. A 1% agarose gel was prepared by adding 1% agarose (w / v) and 2% 50×TAE buffer (v / v) (Table 5) to distilled water and heating to dissolve. Samples used were a mixture of 1.5 μL of Smart Ladder (Nippon Gene, Toyama) and 4.5 μL of 10:1 TE buffer, and a mixture of 5 μL of eDNA extract and 2 μL of Loading Dye (Toyobo, Osaka). Agarose gel electrophoresis (100V, 28 min.) was performed using 1×TAE buffer (diluted from 50×TAE) as the electrophoresis buffer (Mupid-exU, Mupid-2plus, ADVANCE, Tokyo). Afterwards, the gels were stained by immersion in a 20,000-fold diluted ethidium bromide solution for 15 minutes, and then washed by immersion in distilled water for 5 minutes. UV light was irradiated onto agarose gels using a transilluminator (ATTO, ATTO Corporation), and the fluorescence intensity of the DNA bands on the agarose was measured using KODAK 1D Image Analysis software (KODAK, Tokyo). A calibration curve was created to determine the amount of DNA relative to the fluorescence intensity of the DNA bands, and the amount of DNA was determined from the fluorescence intensity of the DNA bands in each sample DNA solution based on this curve. The total number of soil bacteria was determined using a calibration curve that converted the amount of eDNA to the total number of bacteria by DAPI staining. The relationship between the quantified amount of eDNA and total bacteria was given by the equation Y = 1.7 × 10⁻⁶. 8 X(R 2 The total number of soil bacteria was calculated using [Y: total number of soil bacteria (cells / g-soil), X: amount of eDNA (μg / g-soil)].

[0048] [Table 3] [Table 4] [Table 5]

[0049] (5) Test plants and pathogens The plant used was Brassica rapa var. perviridis (Takii Seed Co., Ltd., Kyoto). The pathogen used was Plsamodiophora brassicae, which causes clubroot disease by forming galls on the roots of Brassicaceae crops and inhibiting the supply of nutrients and water to the above-ground parts. Infection and disease development experiments for clubroot disease were conducted in an isolated plant factory (on campus). Cultivation was carried out at a room temperature of 23°C, under light conditions / 12 hours and dark conditions / 12 hours.

[0050] (6) Preparation of resting spore suspension Field-derived komatsuna (Japanese mustard spinach) roots infected with clubroot disease and 1.5 times that amount (v / w) of distilled water were added to a mixer and crushed. The mixture was then filtered through a 500 μm mesh sheet. The filtrate was centrifuged (500 rpm, 5 min.) to precipitate the plant material. The supernatant containing the spore suspension was separated and centrifuged (1,000 rpm, 10 min.), after which the precipitate was suspended in distilled water. This procedure was repeated three times to purify the resting spores. The purified precipitate was suspended in distilled water to obtain a resting spore suspension.

[0051] The roots of komatsuna (Japanese mustard spinach) grown in akadama soil one week after sowing were subjected to a suspension of resting spores of root-knot disease (1.0 × 10). 6The roots were immersed in (spores / mL) for 20 minutes. After immersion, the seedlings were transplanted into Hana-chan potting soil (Hanagokoro, Aichi) and cultivated in a plant growing room (23°C, 16 hours under light conditions / 8 hours under dark conditions) for 8 weeks to allow the dormant spores in the roots to fully mature. The roots were removed from the soil and washed with tap water. The stems and unaffected roots of the komatsuna were carefully removed, leaving only the diseased roots. The obtained diseased roots were similarly crushed, and the dormant spores were purified. 1 / 5 modified Hoagland·10mM MES solution (Tables 6 and 7) was added to the precipitate after purification to prepare a suspension of dormant spores of clubroot fungus, which was stored at 4°C. 1 / 5 modified Hoagland·10mM MES solution is a storage buffer that prevents a decrease in the germination rate of dormant spores. The resting spore concentration of the clubroot fungus resting spore suspension was measured using a hemocytometer (Sunlead Glass, Saitama). The prepared resting spore suspension was stained with 0.01% (w / v) methylene blue staining solution (a mixture of 0.25 ml of 0.2 M disodium hydrogen phosphate solution and 99.75 ml of 0.2 M potassium dihydrogen phosphate solution was mixed with 100 ml of 0.02% (w / v) methylene blue aqueous solution, and the pH was adjusted to 4.6 with 0.05 M hydrochloric acid), and micrographs taken under bright-field microscope are shown in Figure 1.

[0052] [Table 6] [Table 7]

[0053] (7) Infection by pathogens Only two types of organic soil and chemical soil made from the base soil in Table 1 were used. After fertilizing the base soil, while adjusting the water content, a spore suspension in which the resting spores of the clubroot pathogen were mixed with water was inoculated into the soil to prepare a resting spore mixed soil (hereinafter referred to as diseased soil). The prepared soil was left standing in a plant growth chamber (23 °C) for one week to stabilize the total bacterial count. Although the control standard for clubroot disease is said to be 3,000 spores / g-soil, in this experiment, a contamination condition with a higher concentration than that was set. 450 ml of the prepared soil was put into each pot, and three seedlings of Komatsuna that had grown for one week from the seeds were planted per pot, and three pots were prepared for each type of soil. After planting, cultivation was carried out in a plant growing chamber (23 °C, light condition 16 h / dark condition 8 h) for four weeks.

[0054] (8) Growth analysis of plants At the fourth week of cultivation, the pot was inverted, and Komatsuna was carefully taken out from the soil. To measure the total bacterial count and pH of the soil, the soil including the rhizosphere soil attached to the roots of Komatsuna was collected, put into a plastic bag with a clip, and stored at 4 °C. The soil remaining on the roots was carefully washed away with running water, and the moisture was absorbed with a Kim towel. Komatsuna was divided into aboveground and underground parts at the leaf axils of the leaves (Figure 2), and the weights of each were measured.

[0055] (9) Evaluation of the incidence of clubroot disease in plants The incidence of clubroot disease of the harvested Komatsuna was evaluated in five grades as follows (Figure 3). Five-grade evaluation <Class0: No symptoms, Class1: Small knots only on lateral roots, Class2: Small knots on the main root, Class3: Medium to large-scale knots on the main root, which may damage the growth of the plant (lateral roots are healthy), Class4: The root system is completely destroyed, and the growth of the plant is affected (the whole root is diseased or decayed)>.

[0056] From the results of the five-grade evaluation, the disease index (DI) was calculated by the following calculation method. 1, 2, 3, 4 are the disease indices by degree, n1~n4 are the number of strains in each class, Nt is the total number of strains, and the higher the DI, the more negative the impact on plant growth. JPEG2026113726000008.jpg1777

[0057] (10) Evaluation of the incidence of clubroot disease by adding wood vinegar to the soil Infection and disease development experiments for clubroot disease were conducted by varying the concentration of wood vinegar added (weight %) relative to the soil, as shown in Table 8. [Table 8] (11) Analysis of soil bacterial diversity and amplification of 16S rRNA genes by PCR The eDNA extract was diluted as appropriate, and the 16S rRNA gene was amplified by PCR using the compositions (Tables 9 and 10) and conditions (Table 11) shown in the table below. Since the PCR amplification efficiency was poor for eDNA extracts derived from chemical soil, 35 cycles were used for eDNA extracts from organic soil and 40 cycles for eDNA extracts from chemical soil. After amplification, the PCR product and a 100 bp DNA Ladder H3 RTU (Gene DireX) were electrophoresed using a 1.5% agarose gel to confirm the presence and length of the amplified DNA fragments. [Table 9] [Table 10] [Table 11]

[0058] (12) Microbial flora analysis by PCR-DGGE A DCode System (Bio-Rad, Hercules) was used for PCR-DGGE analysis. 8% (w / v) polyacrylamide gels with a denaturant concentration gradient of 27.5% to 67.5% were prepared using 40% (w / v) acrylamide / bis-40 solution (Table 12), urea, formamide, 50×TAE buffer, and a gradient former (Bio-Rad, Hercules) (Table 13). After filling the wells of the prepared acrylamide gels with 1×TAE buffer, a solution of 23 μL of PCR product and 10 μL of Loading Dye (Toyobo, Osaka) was applied. 5 μL of DGGE Marker II (NIPPON GENE, Tokyo) was applied to each end of the gel to allow for comparison of bands between different electrophoresis runs and to account for gel distortion. 7.0 L of 1×TAE buffer was placed in an electrophoresis tank, and the water temperature was set to 60°C using an electrophoresis temperature control module (Bio-Rad, Hercules). A sandwich core unit with an acrylamide gel was placed in the electrophoresis tank (Bio-Rad, California), and electrophoresis was performed by applying current using a power supplier (Power Pac, Bio-Rad, Hercules) (60°C, 15 hr, 70V). After electrophoresis, the acrylamide gel was immersed in distilled water for 30 minutes to elute the denaturant into the distilled water. Subsequently, the gel was stained with ethidium bromide diluted 5,000 times with distilled water (30 min.), and then UV irradiation was performed using a transilluminator (ATTO, ATTO Corporation, Tokyo) to confirm the DNA bands. [Table 12] [Table 13]

[0059] 2.Results (1) Changes in soil bacterial count To compare the incidence of clubroot disease in chemical and organic soil environments, chemical and organic soils were prepared. Both the chemical and organic soils were created by applying chemical or organic fertilizers to woody soils of the same composition. Subsequently, clubroot fungi were introduced into each soil at a rate of 1.0 × 10⁻⁶.5 Diseased soil containing spores / mL-soil was prepared. The total bacterial count of each soil sample was examined, and the total bacterial count of the chemically prepared soil was below the detection limit (6.6 × 10⁻⁶). 6 While the number of cells / g-soil was less than 50%, a significant increase in soil bacteria was observed in organic soil (Figure 4).

[0060] (2) Growth of Komatsuna in diseased soil Komatsuna (Japanese mustard spinach) was cultivated for four weeks in chemical soil and organic soil inoculated with clubroot pathogen. Figure 5 shows the growth of komatsuna in each soil type. The above-ground weight of komatsuna cultivated in chemical soil (non-pathogenic soil) was 11.6g / pot, while the above-ground weight of komatsuna cultivated in chemical soil (pathogenic soil) was 2.18g / pot. Inoculation of the soil with clubroot pathogen reduced the growth of komatsuna by 84%, indicating significant growth inhibition.

[0061] On the other hand, the above-ground weight of komatsuna grown in organic soil (non-pathogenic soil) was 26.8g / pot, while the above-ground weight of komatsuna grown in organic soil (pathogenic soil) was 23.9g / pot, representing only an 11% decrease. This suggests that while inoculation with clubroot fungus slightly reduced the above-ground weight, komatsuna grown in organic soil (pathogenic soil) did not experience the same decline in growth as komatsuna grown in chemical soil (pathogenic soil).

[0062] (3) Komatsuna roots in diseased soil Komatsuna (Japanese mustard spinach) was cultivated in chemical soil (sick soil) and organic soil (sick soil), and the roots of komatsuna infected with clubroot disease were observed. In the komatsuna cultivated in chemical soil (sick soil), almost no lateral roots remained, and clubroots were clearly visible in all plants. In addition, blackened and rotten roots were also observed.

[0063] On the other hand, many komatsuna roots grown in organic soil (sick soil) retained their lateral roots, and some plants showed no signs of the disease. The degree of root-knot disease in these plants suggested that the growth of the komatsuna was significantly affected (Figure 6).

[0064] (4) Disease incidence in diseased soils of chemical and organic soils Komatsuna (Japanese mustard spinach) was cultivated in diseased chemical soil and organic soil, and its roots were observed. After classifying them according to the degree of disease, the disease severity was calculated. In two experiments, the disease severity of komatsuna cultivated in organic soil (disease soil) was lower than that of komatsuna cultivated in chemical soil (disease soil) (Figure 7). This clearly shows that an organic soil environment reduces the infection and onset of clubroot disease. Since the infection and onset of clubroot disease were significantly suppressed in organic soil, which has a high amount of organic matter and a high number of bacteria, it is possible that the abundant bacteria in the soil suppressed the infection and onset of clubroot disease.

[0065] (5) Effects of wood vinegar on diseased soils in chemical and organic soils (i) Growth of Komatsuna Using vermiculite-based soil, diseased soils were prepared by inoculating them with clubroot fungi in chemical and organic soils. To investigate the effect of wood vinegar on clubroot, wood vinegar was added to each diseased soil, and komatsuna (Japanese mustard spinach) was cultivated. Figures 8 and 9 show the growth of the komatsuna and the above-ground fresh weight per pot.

[0066] In chemically treated soil, komatsuna grown in diseased soil grew less vigorously than komatsuna grown in untreated soil (soil without inoculation with clubroot fungus), and the above-ground weight was lower compared to the untreated soil. The above-ground weight gradually increased with the addition of wood vinegar, and increased with increasing amounts of wood vinegar (Figure 8).

[0067] Similarly, in organic soil, when komatsuna was cultivated in diseased soil, the above-ground weight decreased compared to untreated soil, but it recovered after the addition of wood vinegar (Figure 9). These findings suggest that the addition of wood vinegar suppresses the development of clubroot disease.

[0068] (ii) Komatsuna roots and disease severity The underground parts of komatsuna were observed and the degree of disease was measured. In chemical soil, the onset of clubroot disease was significant in diseased soil, with a disease severity of 100. However, when 0.1% wood vinegar was added, the disease severity decreased to 71, and when 0.5% was added, it decreased to 25 (Figure 10).

[0069] In organic soil, the disease severity was 33, but the addition of wood vinegar further reduced the severity, from 21 at a wood vinegar concentration of 0.1% to 4.2 at a concentration of 0.5% (Figure 11). These results clearly show that the addition of wood vinegar to the soil suppresses the infection and occurrence of clubroot disease.

[0070] (iii) Analysis of soil microbiota using PCR-DGGE The soil bacterial flora of cultivated soil was analyzed using the PCR-DGGE method. The band pattern of the soil bacterial flora obtained by PCR-DGGE is shown in Figure 12. In the figure, "M" represents DGGE maker II.

[0071] In both chemically treated and organic soils, inoculation with clubroot fungus significantly altered the band pattern compared to the control group (lanes 1 and 5) (lanes 2 and 6). Conversely, the group treated with wood vinegar (lanes 3, 4, 7, and 8) exhibited almost the same band pattern as the control group. This indicates that the addition of wood vinegar to the soil restores the composition of the soil bacterial flora.

Claims

1. A composition for preventing the onset of clubroot disease in plants, Contains wood vinegar, composition.

2. A composition for improving soil used in plant cultivation that is contaminated with clubroot fungus, Contains wood vinegar, composition.

3. A composition according to claim 1 or 2, The aforementioned plant is a member of the Brassicaceae family. composition.

4. The composition according to claim 3, The aforementioned Brassicaceae plant is a Brassicaceae vegetable. composition.

5. The composition according to claim 4, wherein the cruciferous vegetable is a vegetable selected from the group consisting of Chinese cabbage, cabbage, Brussels sprouts, mizuna, komatsuna, turnip, broccoli, cauliflower, bok choy, nozawana, tsukena, nabana, tatsoi, rapeseed, mustard greens, takana, watercress, radish, and kale. composition.

6. A composition according to any one of claims 1 to 5, The composition is applied to soil for plant cultivation such that the concentration of wood vinegar in the soil is 0.05 to 5.0 w / w%. composition.

7. The composition according to claim 6, The aforementioned soil is soil that does not contain organic fertilizers. composition.

8. A method for preventing the onset of root-knot disease in plants, The process includes the step of applying wood vinegar to the soil in which the plant is being cultivated, or to the soil in which the plant is being cultivated. method.

9. A method for improving soil contaminated with clubroot fungus, The step includes applying wood vinegar to the soil contaminated with the aforementioned clubroot fungus. method.

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