Clubroot mitigating agent and clubroot mitigating material

Abstract

To provide a new chemical substance having germination induction effect on dormant spores of clubroot fungus which is a pathogen of clubroot, and to provide a material containing the new chemical substance.SOLUTION: Ascorbic acid or a related substance of the ascorbic acid subjected to a chemical reaction induces germination of dormant spores causing clubroot. The clubroot reduction material contains the ascorbic acid or a related substance of the ascorbic acid subjected to a chemical reaction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to the reduction and control of clubroot disease in cruciferous vegetables. [Background technology]

[0002] Brassicaceae vegetables, including designated vegetables such as cabbage and Chinese cabbage, are produced in large quantities domestically and are major crops that support Japanese agriculture (Table 1, Ministry of Agriculture, Forestry and Fisheries Crop Statistics for 2019).

[0003] [Table 1]

[0004] In many production sites, cost-first chemical fertilizer cultivation and excessive continuous cropping have led to imbalances in the physical, chemical, and microbial properties of the soil, which in turn trigger the outbreak of various soil diseases. In particular, damage caused by clubroot disease of cruciferous vegetables, which is caused by continuous cropping of cruciferous vegetables, is increasing, and is one of the causes of reduced yields (Table 2, Ministry of Agriculture, Forestry and Fisheries Plant Protection Annual Report for 2014).

[0005] [Table 2]

[0006] Clubroot disease of cruciferous vegetables is a soil-borne disease caused by the obligate parasitic pathogen Plasmodiophora brassicae (Japanese name: clubroot fungus). This fungus exists primarily in the soil as durable resting spores. When it senses the presence of a host, it germinates into zoospores, which then multiply within the host cells through a two-stage infection process—root hair infection and cortical infection—leading to the formation of root clubs. Furthermore, the fungus produces large numbers of resting spores within the galls, which disperse into the soil as the galls decay and disintegrate, becoming a source of infection for the following crop. Infected plants begin to wilt aboveground due to root dysfunction, and in severe cases, they die (Kageyama and Asano, 2009).

[0007] Due to the nature of their "production areas," many of Japan's production areas tend to cultivate the same crop repeatedly. In such fields with continuous cropping, repeated outbreaks of clubroot lead to an increase in the density of dormant spores of the clubroot fungus in the soil over time, posing a problem of reaching a level that is difficult to control with current countermeasures alone. While measures to control clubroot include the introduction of resistant varieties, soil pH adjustment, and the use of chemical pesticides, the emergence of strains of fungi that can overcome resistance has led to an increased reliance on chemical pesticides.

[0008] Chemical pesticides used to control clubroot have the effect of bacteriostatically controlling the resting spores of the clubroot fungus and killing the zoospores, but there is also a risk of producing drug-resistant clubroot fungus. Furthermore, bacteriostatic action does not lead to a reduction in the density of bacteria in the soil, so it has not yet reached a fundamental solution to the disease problem. In addition, in production areas near cities, fields are often adjacent to residential areas, making their use difficult. For these reasons, there is a need to create a control system that does not rely excessively on chemical pesticides.

[0009] Because this fungus is obligately parasitic, it is believed that inducing germination of dormant spores in the absence of a host plant and breaking the infection cycle can effectively reduce the fungal population in soil (White, 1954). Plants used in the hope of inducing germination are called "decoy plants," and their use has long been attempted in Japan (Murakami et al., 2001; Tsushima, 2000; Yamada et al., 1997). However, their adoption is limited due to overlapping crop seasons between the decoy plants and the actual crop, as well as the labor and financial burden of cultivating and managing the decoy plants, which do not contribute to harvest.

[0010] Another possible method for inducing germination of dormant spores in soil in the absence of a host plant is the use of germination inducers. This method offers a significant advantage to growers, as it does not require the use of decoy plants or other cultivation techniques. Previous research has shown that compounds such as caffeic acid, catechin, and coumaric acid promote the germination of dormant spores of Plasmodiophora root-knot fungi (Ohi et al., Biosci. Biotechnol. Biochem. 67(1), 170-173, 2003). Methods for controlling clubroot infection using these substances have been explored (Patent Document 1), but they have not yet been put to practical use. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] Japanese Patent Application Laid-Open No. 2000-128708 Summary of the Invention [Problem to be solved by the invention]

[0012] The present invention aims to propose a new chemical substance that has the effect of inducing the germination of resting spores of Plasmodiophora brassicae, the pathogen of clubroot disease, and a material containing the new chemical substance. [Means for solving the problem]

[0013] [1] Ascorbic acid or a chemically reacted ascorbic acid-related substance that induces germination of resting spores that cause clubroot disease.

[0014] [2] [1] A material for reducing clubroot disease, comprising ascorbic acid or a chemically reacted substance related to said ascorbic acid.

[0015] By directly contacting the resting spores with ascorbic acid, which induces germination of the dormant spores that cause the above-mentioned clubroot disease, or a chemically reacted ascorbic acid-related substance, germination can be induced and the severity of clubroot disease can be reduced.

[0016] [3] A material for reducing clubroot disease, comprising a carrier material to which ascorbic acid [1] or a chemically reacted substance related to said ascorbic acid has been added or mixed.

[0017] [4] A material for reducing clubroot disease, comprising a granular or particulate carrier material coated with ascorbic acid [1] or a chemically reacted substance related to said ascorbic acid.

[0018] Examples of the carrier material include clay minerals such as zeolite and diatomaceous earth, organic materials such as livestock manure compost and castor meal, and ash materials such as wood ash and rice husk ash.

[0019] In order to increase the opportunity for ascorbic acid or chemically reacted ascorbic acid-related substances that induce the germination of the dormant spores that cause the above-mentioned clubroot disease to come into direct contact with the dormant spores, the ascorbic acid or chemically reacted ascorbic acid-related substances that induce the germination of the dormant spores that cause the above-mentioned clubroot disease can be made into a liquid or granular material, and its concentration can be 0.5-20% (w / w).

[0020] The above-mentioned effects can be achieved by applying a 10-fold diluted solution or the undiluted solution once at a rate of 40 l / 10 a. [Effects of the Invention]

[0021] According to this invention, it is possible to provide a novel chemical substance that has the effect of inducing the germination of resting spores of Plasmodiophora brassicae, the pathogen of clubroot disease, and a material containing said novel chemical substance. By introducing the novel chemical substance with the germination-inducing effect as a "decoy material," it is possible to provide a system for controlling clubroot disease. [Brief explanation of the drawings]

[0022] [Figure 1] Graph showing the incidence of clubroot disease caused by treatment with each organic acid. [Figure 2] A graph showing the severity of clubroot disease depending on the treatment conditions for each organic acid and resting spore. [Figure 3] A graph showing the severity of clubroot disease under treatment conditions of various concentrations of ascorbic acid and resting spores. [Figure 4] A graph showing the killing effect of dormant spores by one hour of treatment with ascorbic acid. [Figure 5] A graph showing the effect of long-term ascorbic acid treatment on killing dormant spores. [Figure 6] A diagram showing the results of a survey on the incidence of clubroot disease on flower vegetables following the application of materials containing new chemical substances that have the effect of inducing the germination of resting spores. [Figure 7] A graph showing the results of confirming the occurrence of clubroot disease using soil before and after application of a material containing a new chemical substance that has the effect of inducing the germination of resting spores. [Figure 8] Reference photograph showing the solubility of ascorbic acid solution and the presence or absence of precipitation after cooling overnight in a storage test. [Figure 9] Reference photograph showing the solubility of ascorbic acid solution and the presence or absence of precipitation after 30 days of storage in a storage test. DETAILED DESCRIPTION OF THE INVENTION

[0023] <Selection of germination inducers for resting spores of Plasmodiophora brassicae> A substance (i.e., a germination inducer) having an effect on inducing germination of resting spores of Plasmodiophora brassicae (i.e., resting spores of Plasmodiophora brassicae) was selected as follows.

[0024] In order to select a germination inducer for Plasmodiophora root-knot pathogen dormant spores, we examined the following candidate chemicals: coumaric acid, caffeic acid, and catechin (5 mM each), which have already been reported to be effective; glutathione (5 mM), L-ascorbic acid, sodium glutamate, and sodium 5'-ribonucleotides (0.1% each).

[0025] Resting spores of Plasmodiophora root canaliculata were added to each organic acid solution, and 2.5 × 10 5 A 20 ml solution containing 1000 cells / ml was prepared and allowed to stand at 24°C for one week.

[0026] Then, 20 ml of each solution was added to 50 g of sterilized Nippi horticulture soil No. 1, and the mixture was left to stand at 24°C under dry conditions for 2 weeks.

[0027] Four replicates of the treated soil were diluted 16-fold with Nippi Engei Soil No. 1, and each treated soil was filled into five cells of a cell tray. Two plants of the clubroot-infected flower vegetable variety 'Hana Kazari' were sown per cell, and a cell tray test was conducted.

[0028] In addition, 20 ml of a mixed solution of each organic acid solution and resting spores was added to sterilized Nippi horticultural soil No. 1 immediately after preparation and left to stand at 24°C under dry conditions for 3 weeks, and the same study was conducted.

[0029] The effect of each organic acid on the onset of clubroot disease is shown in Figure 1. Direct application of coumaric acid, caffeic acid, catechin, and L-ascorbic acid was found to have a disease-suppressing effect.

[0030] Therefore, we examined the disease suppression effects of organic acid treatment followed by inoculation into the culture soil or direct inoculation into the culture soil for caffeic acid, catechin, and L-ascorbic acid, which have high potential for use as fertilizer ingredients (Figure 2).

[0031] When resting spores of Plasmodiophora root-knot fungus were inoculated into the soil after being treated with an organic acid solution for one week, caffeic acid, catechin, and L-ascorbic acid significantly suppressed the onset of clubroot disease.

[0032] On the other hand, when the organic acid solution containing resting spores was directly inoculated into the culture medium, no clear disease suppression effect was observed. This is presumably because the buffering effect of the culture medium reduced the effect of the organic acid solution.

[0033] <Conditions for ascorbic acid suppression of clubroot disease> Resting spores of Plasmodiophora root canal were added to L-ascorbic acid solutions (0%, 0.1%, 0.5%, and 2.5%), and 2.5 × 10 5 A 20 ml mixture solution of 1000 / ml was prepared.

[0034] <Test A> The mixed solution was allowed to stand at 24°C for one week, then added to 50 g of sterilized Nippi horticulture soil No. 1, and allowed to stand at 24°C under dry conditions for two weeks.

[0035] <Test B> Immediately after preparation, the mixed solution was added to 50 g of sterilized Nippi horticulture soil No. 1 and allowed to stand at 24°C under dry conditions for 3 weeks.

[0036] <Test C> Immediately after preparation, the mixed solution was added to 15 g of sterilized Nippi horticultural soil No. 1 and allowed to stand at 24°C for 1 week. After that, 35 g of sterilized Nippi horticultural soil No. 1 was added and allowed to stand at 24°C under dry conditions for 2-4 weeks.

[0037] Two replicates of each treatment soil were diluted 25-fold with Nippi Engei Soil No. 1 and filled into 10 cells of a cell tray. Two plants of the clubroot-infected flower vegetable variety 'Hanazakazari' were sown per cell and a cell tray test was conducted.

[0038] <Results of the study> The effect of L-ascorbic acid on the development of clubroot disease under each treatment condition is shown in Figure 3.

[0039] When L-ascorbic acid solutions of various concentrations and resting spores were directly treated, a high disease suppression effect was observed at all concentrations.

[0040] When a mixed solution of L-ascorbic acid containing resting spores was directly inoculated into the culture soil, the disease suppression effect was observed under normal moisture conditions and under flooded conditions, and the effect improved significantly as the L-ascorbic acid concentration increased.

[0041] This indicates that even if L-ascorbic acid solution is directly inoculated into the soil, the disease can be suppressed as long as the amount of water added is just enough to moisten the soil. It is also thought that a higher concentration of L-ascorbic acid increases the probability of contact with dormant spores, making it more effective.

[0042] <Confirmation of the bactericidal effect of ascorbic acid on clubroot dormant spores> To prove that ascorbic acid suppresses clubroot disease and is not classified as a pesticide, we confirmed whether ascorbic acid has a bactericidal effect on resting spores by determining whether the resting spores are viable or not using Evans blue staining.

[0043] At Kyoto Prefectural University, we performed a short-term test to determine the viability of resting spores of Plasmodiophora brassicae after treatment with various concentrations of ascorbic acid. Five microliters of Plasmodiophora brassicae resting spores were added to 500 μl of L-ascorbic acid solution (0%, 0.1%, 0.5%, 2.5%), resulting in a final concentration of 1 × 10 7 The mixture was made into a solution of spores / ml and allowed to stand at 24°C for 1 hour (triplicate).

[0044] As a control, the resting spore solution was left to stand at 95°C for 1 hour to form a heat-treated group. 100 μL of each treatment solution was mixed with an equal volume of Evans blue solution and left to stand at 24°C in the dark for 24 hours for staining. An equal volume of sterile water was added to the staining solution, and the unstained spores (live spores) and stained spores (dead spores) were counted under an optical microscope, and the stained spore rate was calculated as (number of unstained spores + number of stained spores) / total number of spores.

[0045] In an in-house test at Asahi Agria Co., Ltd., the viability of dormant spores was determined after one week of L-ascorbic acid treatment.

[0046] One ml of a suspension of Plasmodiophora brassicae dormant spores (derived from infected Komatsuna plants in Saitama Prefecture) prepared in sterile water was added to a test tube with 1 ml of L-ascorbic acid solution (0, 0.2, or 2.0% (w / v)) prepared in 20 mM HEPES buffer (pH 7) or sterile water. The mixtures were then mixed in duplicate to obtain final L-ascorbic acid concentrations of 0, 0.1, or 1.0% (w / v). The mixtures were shaken at 60 reciprocations per minute at 25°C to prevent the dormant spores from settling or forming clumps. On days 1, 5, and 14, 150 μl aliquots were transferred to plastic tubes. A 50 μl aliquot of the 150 μl was counted using a hemocytometer. A 100 μl aliquot was harvested, washed three times with 10 mM HEPES buffer (pH 7), suspended in 50 μl, and mixed with an equal volume of 10% Evans Blue stain. After 15 minutes, the number of unstained and stained spores was counted under a light microscope, and the percentage of dead spores was calculated.

[0047] <Bactericidal effect of ascorbic acid treatment on resting spores of Plasmodiophora root-knot fungus> The effects of L-ascorbic acid on dormant spores are shown in Figures 4 and 5.

[0048] The dormant spores were not killed by treatment with L-ascorbic acid for 1 hour.

[0049] In addition, for the long-term ascorbic acid treatment, counting the total number of bacteria on the 15th day was abandoned due to the formation of clumps in the test solution, but the number of bacteria did not decrease rapidly after 5 days of L-ascorbic acid treatment.

[0050] The dead spore rate increased over the 14th day in the 0% L-ascorbic acid group, whereas the L-ascorbic acid-treated group remained stable or showed a decreasing trend.

[0051] The increase in the percentage of dead spores in the 0% L-ascorbic acid group is thought to be due to the death of dormant spores over time in the solution.

[0052] These results indicate that L-ascorbic acid does not have a significant decomposition or bactericidal effect on dormant spores.

[0053] [Table 3]

[0054] <Ascorbic acid solution solubility, storage test> L-ascorbic acid was added to water heated to 50°C and stirred with a stirrer. After confirming that the L-ascorbic acid had dissolved, the mixture was stirred for an additional 10 minutes or more. After cooling at room temperature overnight, the presence or absence of precipitation was confirmed, and the pH and specific gravity of the solution were measured. The solution was dispensed into three containers and left to stand at room temperature, 4°C, and 35°C to confirm its storage stability.

[0055] [Table 4]

[0056] result When 10-50% (w / w) L-ascorbic acid solutions were prepared, all were soluble. After standing overnight, solutions with concentrations of 35% or higher formed crystals or precipitates (Table 5, Figure 8). Solutions with concentrations of 10-30% were colorless, transparent, or slightly yellowish. The pH was around 2, decreasing as the concentration increased.

[0057] [Table 5]

[0058] L-ascorbic acid solutions that did not produce crystals or precipitates were left standing at room temperature, 4°C, and 35°C. The 25-30% solutions produced crystals at room temperature and 4°C, while the 20% solution produced crystals at 4°C. Furthermore, the ascorbic acid solutions stored at room temperature began to change color after 20 days, and after 3 days at 35°C, and turned yellow to brown by the 30th day (Figure 9).

[0059] <Development of candidate materials containing new chemical substances that induce germination of clubroot dormant spores> Liquid and pellet materials were prepared as materials that are expected to be used in fields contaminated with clubroot in the future.

[0060] For the liquid material, a 20% (w / w) solution of L-ascorbic acid was prepared.

[0061] We also created pellets containing coffee grounds containing caffeic acid and L-ascorbic acid. Quail compost and air-dried coffee grounds were mixed in a 6:4 weight ratio, the moisture content adjusted to 30% of the maximum water capacity, and the mixture was filled into a 10-liter fermentation tank.

[0062] The mixture was mixed and the moisture content adjusted once a week for four weeks while fermenting to make coffee grounds compost.

[0063] Coffee ground pellets and coffee ground pellets containing L-ascorbic acid were granulated in the blending ratios shown in Table 6 below.

[0064] [Table 6]

[0065] <Field test of clubroot disease on flower vegetables using a germination inducer for clubroot resting spores> In paddy field No. 12 at the Kyoto Prefectural Agriculture, Forestry and Fisheries Technology Center experimental field, where clubroot disease had been confirmed, L-ascorbic acid solution (20% (w / w)), coffee grounds pellets, and coffee grounds pellets containing L-ascorbic acid were applied, with no treatment and treatment with Oracle powder serving as controls.

[0066] L-ascorbic acid solution (20% (w / w)) 500 g / m 2 Dilute 5 times with water to give a diluted solution of 2.5 l / m 2 (equivalent to 100 kg / 10 a of L-ascorbic acid) was sprayed, and the L-ascorbic acid solution-treated area was covered with mulch to prevent runoff due to rainfall.

[0067] The L-ascorbic acid solution treatment was sprayed once on August 6th, and three times on July 28th, August 3rd, and August 6th.

[0068] Coffee ground pellets and coffee ground pellets with L-ascorbic acid were sold at 200 g / m on August 6th.2 (equivalent to 200 kg / 10 a) was applied.

[0069] Oracle dust was applied on August 7. The cultivated variety was 'Hanazakaze', a flowering vegetable variety affected by clubroot disease.

[0070] There were three rows per ridge, and to prevent damping-off disease, each row was sown the first time (August 18-19), planted (August 25), and sown the second time (September 1). 2 , performed in triplicate.

[0071] A root-knot disease survey was conducted for the first sowing on October 1st and the second sowing on October 12th.

[0072] Ten plants were examined for each treatment area, and plants that could not be evaluated due to damping-off or broken roots were excluded, and the remaining plants were examined in the same way.

[0073] <Cell tray test> After removing plant debris and stones from the air-dried test soil, the soil was crushed with a hammer and sieved to a 2 mm sieve. The soil was mixed with Nippi horticultural soil No. 1 in a 1:4 ratio and filled into a 25-well cell box. Seeds of the flower vegetable cultivar 'Hana Kazari' were sown and cultivated using the bottom watering method. One month after sowing, the extent of root disease was evaluated (cell tray assay, Yoshimoto et al., 2001).

[0074] The severity of clubroot disease was evaluated based on the evaluation method of Kuginuki et al. (1999) using four levels: 0: no nodules, 1: small nodules on lateral roots, 2: between 1 and 3, and 3: large nodules on main and lateral roots. The severity was calculated using the following formula.

[0075]

number

[0076] Soil samples were collected from each treatment area before (July 28) and after (August 18) application of the materials, and used for cell tray tests and to measure the density of Plasmodiophora root-knot fungi in the soil. The density of Plasmodiophora root-knot fungi was measured by the LAMP method under contract.

[0077] In the LAMP method, 0.4 g of sieved test soil is heated and mixed in a buffer, and after adding a binding buffer and 2-propanol, DNA is purified using a spin column. The purified DNA is added to the LAMP reaction reagent, and the amplification of Plasmodiophora root-knot fungus DNA is measured using a turbidimeter, and the number of Plasmodiophora root-knot fungi is quantified from the amplification results.

[0078] <Field test of clubroot disease on flower vegetables using materials containing new chemical substances that induce germination of clubroot resting spores> The incidence of clubroot disease on flower vegetables due to application of materials is shown in Figure 6.

[0079] All plants showed good initial growth, but in the first survey many plants showed wilting symptoms and many roots were torn off (results not shown).

[0080] In the second survey, fewer plants showed damping-off symptoms. The disease severity was low overall, and the differences between treatments were unclear, but the plots treated with L-ascorbic acid three times and the plots treated with coffee grounds pellets containing L-ascorbic acid showed a tendency to suppress the disease compared to the untreated plot.

[0081] In addition, this year's rainfall was lower than usual, making conditions less favorable for clubroot disease to develop, and since Oracle treatment did not have any effect in suppressing the disease, it is thought that the materials were not effective.

[0082] <Field test (cell tray test) of clubroot disease on flower vegetables using materials containing new chemical substances that induce germination of clubroot dormant spores> In the field test, the amount of rainfall was lower than usual, which may have affected the severity of clubroot disease.

[0083] Therefore, cell tray tests and soil bacterial density were measured using soil samples collected before (July 28th) and after (August 18th) application of the materials, and the results are shown in Figure 7.

[0084] The application of each material had no effect on soil pH. Furthermore, there was no significant decrease in the density of Plasmodiophora root-knot fungi in any of the treatments, but the disease severity was significantly lower in the three-times L-ascorbic acid treatment plot than in the untreated plot (U test, 5% level).

[0085] When comparing before and after treatment with the materials, the disease severity in the areas treated with L-ascorbic acid three times, coffee grounds pellets, and coffee grounds pellets containing L-ascorbic acid was significantly lower than before treatment (U test, 1% level).

[0086] From this, it is thought that L-ascorbic acid exerts a disease suppression effect when applied multiple times.

[0087] No correlation was found between the density of Plasmodiophora root in the soil before and after treatment and the severity of the disease. This may be due to the difference in conditions between the L-ascorbic acid-treated area and the other treatment areas, which were covered with mulch during treatment, and the movement of resting spores due to the flow of water in the soil down the gradient.

[0088] Based on the above results, although it depends on the weather and soil conditions in the field, it is expected that L-ascorbic acid solution will be effective in reducing clubroot disease, and it is thought that multiple treatments of L-ascorbic acid will increase contact with the clubroot disease fungus in the soil, thereby suppressing the onset of the disease.

[0089] All of the above-mentioned studies were conducted using L-ascorbic acid, the L-isomer of ascorbic acid. Based on the results of these studies, it is believed that ascorbic acid itself and related substances of ascorbic acid that have undergone chemical reactions, such as L-ascorbic acid, can all exert the effects of inducing the germination of dormant spores of Plasmodiophora root-knot fungus, suppressing the onset of Plasmodiophora root-knot disease, and reducing Plasmodiophora root-knot disease, as confirmed in the above-mentioned studies.

[0090] In addition to L-ascorbic acid, chemically reacted ascorbic acid-related substances include ascorbic acid radical, dehydroascorbic acid (DHA), 2,3-diketogulonic acid (DKG), reductic acid, L-xylosone, 3-keto-4-deoxypentosulose (KDP), oxalic acid, and L-threonic acid, which are substances produced from ascorbic acid through chemical reactions caused by factors such as temperature and moisture.

Claims

1. Ascorbic acid or a chemically reacted ascorbic acid-related substance that induces germination of resting spores that cause clubroot disease.

2. A material for reducing clubroot disease, comprising the ascorbic acid according to claim 1 or a chemically reacted ascorbic acid-related substance.

3. 10. A material for reducing clubroot disease, comprising a carrier material to which the ascorbic acid or a chemically reacted substance related to said ascorbic acid as defined in claim 1 has been added or mixed.

4. 2. A material for reducing clubroot disease, comprising a granular or particulate carrier material coated with the ascorbic acid of claim 1 or a chemically reacted substance related to said ascorbic acid.

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