Spore germination inducer and induction method for diaporthe destruens

Abstract

To provide means for inducing spore germination in Diaporthe destruens.SOLUTION: The present invention provides a spore germination inducer for Diaporthe destruens, containing D(+)-fucose or jalapin as an active ingredient, and a spore germination induction method for Diaporthe destruens, including the step of contacting the spore germination inducer with spores of Diaporthe destruens.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a spore germination inducer for sweet potato base rot fungus. The present invention also relates to a method for inducing spore germination in sweet potato base rot fungus using the spore germination inducer, a method for controlling sweet potato base rot disease, a method for screening for a substance that inhibits spore germination induction in sweet potato base rot fungus, and a method for detecting sweet potato base rot fungus. [Background technology]

[0002] Sweet potato root rot is a soil-borne disease caused by the filamentous fungus Diaporthe destruens. Infected sweet potatoes first show brownish black spots at the base of the stem, then the symptoms progress to the above-ground and underground parts, adversely affecting growth and causing poor growth. In severe cases, the plant dies and the sweet potato cannot be harvested. The first outbreak of this disease in Japan was confirmed in Okinawa and Kagoshima prefectures in 2018, and it has now been confirmed to occur in almost all sweet potato producing areas in Japan, including major sweet potato producing areas. Proposed measures to control this disease include the use of resistant varieties, drainage measures for fields, and the use of chemicals. However, in order to effectively prevent the damage and spread of this disease, it is necessary to develop further control methods based on the characteristics of the pathogen and to take comprehensive control measures that combine these methods.

[0003] Spore germination of this pathogen is the initial infection behavior on a plant body, and establishing a method for controlling it could lead to the development of new control materials and methods. However, the physiological characteristics of this pathogen have not been fully elucidated, and currently no method has been established for inducing spore germination of this pathogen. It is known that spore germination can be induced in other filamentous fungi different from this pathogen by sugar components (Non-Patent Documents 1 and 2) or plant excretions (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Osherov N, May G (2000) Conidial germination in Aspergillus nidulans requires RAS signaling and protein synthesis. Genetics 155:647-656. [Non-Patent Document 2] Blakeman JP (1975) Germination of Botrytis cinerea conidia in vitro in relation to nutrient conditions on leaf surfaces. Trans.Br.Mycol.Soc.65: 239-247. [Non-Patent Document 3] Schroth MN, Hildebrand DC (1964) Influence of plant exudates on root-infecting fungi. Annu.Rev.Phytopathol. 2:101-132. Summary of the Invention [Problem to be solved by the invention]

[0005] It is known that most filamentous fungal spores generally germinate in normal nutrient media, and the filamentous fungal taxonomic group to which the sweet potato base rot fungus belongs has the same characteristics. However, the identification of spore germination-inducing components under actual sweet potato cultivation environmental conditions, and the conditions for synchronously germinating spores of the sweet potato base rot fungus by adding such components, have not been elucidated. The present invention has been made under such a background, and aims to provide a means for inducing spore germination of sweet potato base rot. [Means for solving the problem]

[0006] Means for Solving the Problems The present inventors have conducted extensive research to solve the above problems and have discovered that D(+)-fucose induces spore germination of the pathogenic fungus Causing root rot of sweet potato, thereby completing the present invention. That is, the present invention provides the following [1] to [8].

[0007] [1] A spore germination inducer for sweet potato root rot fungus, which contains D(+)-fucose or yarapin as an active ingredient.

[0008] [2] A method for inducing spore germination of sweet potato root rot fungus, comprising a step of contacting the spore germination inducer of sweet potato root rot fungus described in [1] with spores of sweet potato root rot fungus.

[0009] [3] A method for controlling sweet potato root rot, comprising the following steps (1) and (2): (1) A step of applying the spore germination inducer of sweet potato root rot fungus described in [1] to a control object to germinate spores of sweet potato root rot fungus present in the control object; (2) After step (1), a step of applying a control agent for sweet potato root rot to the control target.

[0010] [4] The method for controlling sweet potato root rot according to [3], characterized in that the object to be controlled is derived from a field in which a plant of the Convolvulaceae family is cultivated.

[0011] [5] The method for controlling sweet potato root rot according to [3], wherein the object to be controlled is derived from a field in which sweet potatoes are grown.

[0012] [6] A method for screening for a spore germination induction inhibitor of sweet potato root rot fungus, comprising the following steps (1) and (2): (1) contacting a spore germination inducer for sweet potato root rot fungus described in [1] with spores of sweet potato root rot fungus in the presence of a test substance; (2) After step (1), a step of observing the germination state of spores of the sweet potato root rot fungus.

[0013] [7] A method for detecting sweet potato root rot fungus, comprising the following steps (1) and (2): (1) A step of contacting a sample suspected of containing sweet potato root rot fungus with the spore germination inducer of sweet potato root rot fungus described in [1]; (2) after step (1), a step of detecting sweet potato root rot fungus.

[0014] [8] The method for detecting sweet potato root rot fungus according to [7], characterized in that the sample suspected of containing sweet potato root rot fungus is derived from a plant body of a Convolvulaceae family or a field in which a Convolvulaceae family plant is cultivated. Effect of the Invention

[0015] The present invention provides a novel spore germination inducer for sweet potato base rot fungus. By using the spore germination inducer of the present invention, it is possible to effectively control sweet potato base rot disease, efficiently screen for inhibitors of spore germination induction for sweet potato base rot fungus, and highly sensitively detect sweet potato base rot fungus. [Brief description of the drawings]

[0016] [Figure 1] Photographs showing the germination of spores of the sweet potato root rot fungus on various parts of sweet potato tissue. The arrows indicate the damaged areas. Many germinated spores were observed around the damaged areas (top), whereas no germinated spores were observed around the undamaged areas (bottom). [Diagram 2] Figure showing the spore germination rate of B. cerevisiae in suspensions containing various concentrations of exudate from cut sweet potato stems. The germination rate was counted per field (mean ± sd, n=3) (test of the difference in proportions for all measurements, Ryan's test, α=0.05). [Diagram 3] An optical microscope photograph showing the germination state of spores of the sweet potato root rot fungus. [Figure 4] FIG. 2 is a graph showing the spore germination rate of the pathogenic fungus, Bacillus subtilis, in suspensions containing various concentrations of homogenized sweet potato leaves or stems. [Diagram 5] Figure showing the spore germination rate of the fungus, Causal agent of sweet potato root rot, in suspensions containing various sugar components at different concentrations. Counted in units of 90-110 spores (t-test with no sugar added, n=3, p<0.05). [Figure 6] Figure showing the spore germination rate of the fungus Pseudomonas aeruginosa in suspensions containing various concentrations of D(+)-fucose or L(-)-fucose, counted in units of 90-110 (t-test with no addition, n=3, p<0.05). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described in detail below. The spore germination inducer for sweet potato root rot fungus of the present invention (hereinafter sometimes simply referred to as the ``spore germination inducer of the present invention'') is characterized by containing D(+)-fucose or yarapin as an active ingredient.

[0018] D(+)-fucose is commercially available as a reagent, and such a commercially available product can be used in the present invention. Yarapin is a glycolipid that contains D(+)-fucose as one of its constituent sugars. D(+)-fucose and yarapin may be purified or unpurified. For example, the exudate that comes out from a wound in sweet potato tissue contains D(+)-fucose and yarapin, and the spore germination inducer of the present invention may contain this exudate.

[0019] The spore germination inducer of the present invention can be used for various purposes as described below, and can be used in a form appropriate for the purpose, for example, in the form of an aqueous D(+)-fucose solution.

[0020] The content of D(+)-fucose or jalaphin in the spore germination inducer of the present invention is not particularly limited as long as it is within a range capable of inducing spore germination of sweet potato root rot fungus. For example, when an aqueous solution of D(+)-fucose is prepared, the content of D(+)-fucose can be preferably 0.05 to 5% (w / v), and more preferably 0.1 to 2% (w / v).

[0021] The spores of the sweet potato root rot fungus are classified into alpha spores and gamma spores. The spore germination inducer of the present invention can be used to induce germination of either of these spores, but preferably, alpha spores are used to induce germination.

[0022] The spore germination inducer of the present invention can be applied to, for example, (A) a method for inducing spore germination of sweet potato base rot fungus, (B) a method for controlling sweet potato base rot, (C) a method for screening a substance that inhibits spore germination induction of sweet potato base rot fungus, and (D) a method for detecting sweet potato base rot fungus. These inventions will be described below.

[0023] (A) Method for inducing spore germination of sweet potato root rot fungus The method for inducing spore germination of sweet potato root rot fungus of the present invention is characterized by comprising a step of contacting the spore germination inducer of the present invention with spores of sweet potato root rot fungus.

[0024] The method of contacting the spore germination inducer of the present invention with spores of the sweet potato root rot fungus can be any method depending on the application of the spore germination inducer of the present invention. For example, spores of the sweet potato root rot fungus may be added to a solution containing the spore germination inducer of the present invention, or the spore germination inducer of the present invention may be sprayed on plants, soil, etc. in which spores of the sweet potato root rot fungus are present.

[0025] (B) Method for controlling sweet potato root rot The method for controlling sweet potato base rot of the present invention is characterized by comprising: (1) a step of applying the spore germination inducer of the present invention to an object to be controlled and causing spores of the sweet potato base rot fungus present in the object to germinate; and (2) a step of applying a control agent for sweet potato base rot to the object to be controlled after step (1).

[0026] There is a case in which a spore germination inducer was used to control clubroot disease of cruciferous vegetables (JP Patent Publication No. 2000-128708). Similarly, it is believed that by forcing germination of spores of the sweet potato root rot fungus remaining in fields or seedbeds using the spore germination inducer of the present invention, it is possible to weaken the viability and resistance to chemical treatment, thereby quickly killing the sweet potato root rot fungus.

[0027] The control object is not particularly limited as long as it may contain the sweet potato root rot fungus. The sweet potato root rot fungus infects plants of the Convolvulaceae family, such as sweet potato, so for example, objects originating from fields where plants of the Convolvulaceae family are cultivated can be the control object. "Objects originating from fields where plants of the Convolvulaceae family are cultivated" are, for example, soil and plant residues originating from fields where plants of the Convolvulaceae family are cultivated.

[0028] The spore germination inducer of the present invention to be applied may be an aqueous solution of D(+)-fucose, but like common pesticides, D(+)-fucose or yarapin may be mixed with a carrier, surfactant, dispersant, or adjuvant, and formulated in the usual manner, for example, as a wettable powder, emulsifiable concentrate, dust, or granule.

[0029] The application method is not particularly limited as long as it is a method capable of inducing spore germination of the sweet potato root rot fungus, and examples thereof include spraying, coating, soil drenching, and soil incorporation.

[0030] The amount of the spore germination inducer of the present invention to be applied is not particularly limited as long as it is within a range capable of inducing spore germination of the sweet potato root rot fungus. For example, when D(+)-fucose is used as an active ingredient, it is preferable to apply the agent so that the amount of D(+)-fucose is 10 to 700 kg per 10 ares, and it is more preferable to apply the agent so that the amount is 20 to 300 kg per 10 ares.

[0031] The control agent for sweet potato base rot applied in step (2) is not particularly limited, and commercially available control agents can be used. Currently, G-Fine (registered trademark) wettable powder (active ingredient: anhydrous copper sulfate), Z Bordeaux (active ingredient: basic copper sulfate), Amistar 20 flowable (active ingredient: azoxystrobin), Trifmin wettable powder (active ingredient: triflumizole), etc. are used to control sweet potato base rot, but these may also be used in the present invention. The application amount of the control agent can be appropriately determined depending on the control agent used.

[0032] The timing of application of the control agent for sweet potato base rot is not particularly limited as long as it is after step (1), but it is preferable to apply it when spores of the sweet potato base rot fungus germinate and become mycelium with low viability and drug resistance. Specifically, it is preferable to apply it 6 to 72 hours after application of the spore germination inducer of the present invention.

[0033] (C) Screening method for inhibitors of spore germination induction of sweet potato root rot fungus The screening method for a substance that inhibits spore germination induction of sweet potato root rot fungus of the present invention is characterized by comprising: (1) a step of contacting the spore germination inducer of the present invention with spores of sweet potato root rot fungus in the presence of a test substance; and (2) a step of observing the germination state of the spores of sweet potato root rot fungus after step (1).

[0034] The test substance is not particularly limited and may be either a natural product or a synthetic compound. Specific examples include proteins, peptides, vitamins, hormones, polysaccharides, oligosaccharides, monosaccharides, low molecular weight compounds, nucleic acids (DNA, RNA, oligonucleotides, mononucleotides, etc.), lipids, etc. In addition, the test substance may be purified, but may also be unpurified cell extracts, cell disruption liquids, cell lysates, etc.

[0035] The method of contacting the spore germination inducer of the present invention with spores of the sweet potato root rot fungus is not particularly limited as long as it is a method capable of inducing spore germination of the sweet potato root rot fungus. For example, the spores of the sweet potato root rot fungus may be added to a solution containing the spore germination inducer of the present invention. In this case, the concentration of the spore germination inducer of the present invention in the solution may be preferably 0.05 to 5% (w / v), more preferably 0.1 to 2% (w / v). In addition, the concentration of the spores of the sweet potato root rot fungus in the solution may be preferably 10 3 ~10 7 The concentration can be 10 / mL, and more preferably 10 4 ~10 6 It can be expressed as pieces / mL.

[0036] The timing of observing the spore germination state is not particularly limited as long as it is after step (1), but since a certain amount of time is required for spore germination, it is preferable to observe after such a time has elapsed. Specifically, it is preferable to observe 10 to 30 hours after the contact of the spore germination inducer of the present invention with the spores of the sweet potato root rot fungus.

[0037] The method for observing the germination state is not particularly limited, and can be, for example, observation using a microscope, etc. If the spores do not germinate as a result of the observation, it can be determined that the test substance is a spore germination induction inhibitor, and if the spores germinate, it can be determined that the test substance is not a spore germination induction inhibitor.

[0038] It is possible that the spores do not germinate due to reasons other than the action of the test substance. Therefore, it is desirable to contact the spore germination inducer of the present invention with spores of Pseudomonas aeruginosa in the absence of the test substance, observe the spore germination state in this case, and confirm that the spore germination induction is inhibited by the action of the test substance.

[0039] The spore germination-inducing inhibitors of sweet potato root rot fungus obtained by this screening method will be candidates for new control agents for sweet potato root rot.

[0040] (D) Method for detecting sweet potato root rot fungus The method for detecting sweet potato base rot fungus of the present invention is characterized by comprising: (1) a step of contacting the spore germination inducer of the present invention with a sample suspected of containing sweet potato base rot fungus; and (2) a step of detecting sweet potato base rot fungus after step (1).

[0041] There is a case in which the germination of Pythium spores was promoted and Pythium spores in soil were detected with high sensitivity (JP Patent Publication No. 10-99095). Similarly, it is believed that the spore germination inducer of the present invention can be used to force germination of spores of the sweet potato base rot fungus, temporarily increasing the biomass of the sweet potato base rot fungus and making it possible to detect the sweet potato base rot fungus in the form of spores that are lurking in plant bodies or in the soil with high sensitivity.

[0042] The sample suspected of containing the sweet potato root rot fungus is not particularly limited. Since the sweet potato root rot fungus infects plants of the Convolvulaceae family, for example, the plant body (leaves, petioles, stems, roots, etc.) of a plant of the Convolvulaceae family or something derived from a field where a plant of the Convolvulaceae family is cultivated (soil or plant residue) can be used as the sample.

[0043] The method for contacting the spore germination inducer of the present invention with a sample is not particularly limited as long as it is a method that can induce spore germination of the sweet potato root rot fungus, and examples include methods of spraying, applying, adding, or mixing the spore germination inducer of the present invention with the sample.

[0044] The timing of collecting the sample to detect the sweet potato root rot fungus in the sample is not particularly limited as long as it is after step (1), but it is preferably when the spores present in the sample have germinated and the biomass has increased sufficiently. Specifically, it is preferable to collect the sample 24 to 120 hours after the spore germination inducer of the present invention contacts the spores of the sweet potato root rot fungus.

[0045] The method for detecting the sweet potato root rot fungus in a sample is not particularly limited. For example, the fungus can be detected by a dilution plate method in which a solution obtained by suspending a sample in water is spread on a solid medium, or by quantitative PCR in which the amount of fungal DNA is measured using DNA extracted from a sample. EXAMPLES

[0046] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0047] (1) Experimental method (1-1) Preparation of spore suspension The sweet potato root rot fungus MAFF 246953 strain was cultured on a 0.5x PDA plate medium at 25°C for 4 weeks. Sterile water was added to the bacterial lawn formed on the plate, and the spores on the bacterial lawn were suspended by pipetting so as not to damage the surface of the medium. The suspension was collected in a microtube and washed three times with sterile water by centrifugation (5000xg, 1 min.). 5 x 10 5 A spore suspension was prepared at a concentration of 100 spores / mL.

[0048] (1-2) Observation of spore germination on sweet potato tissue A 5 cm length of stem or petiole or a single leaf was cut from a sweet potato seedling (variety: Beniazuma, using Kumiai Nippi Engei Baido No. 1 as culture soil) that had been grown in an air-conditioned greenhouse for about 3 months at 28°C, and placed on a 9 cm petri dish lined with moist filter paper. A cut of about 5 mm in length was made on the surface of the stem, petiole, or leaf with a razor, and the above-mentioned spore suspension (5 × 10 5 After dropping 3 μL of a spore solution (100 spores / mL) into the dish, the dish was covered with a lid to create a moist chamber, and the dish was left to stand in a dark place at 25°C for 20-24 hours. After standing, the observation site of each tissue was stained with lactophenol cotton blue solution, and the epidermis of the stems and petioles was thinly cut with a razor, and the state of spore germination on each tissue was observed under an optical microscope (BZ-X700, Keyence).

[0049] (1-3) Induction of spore germination by stem exudates The stems of sweet potato seedlings grown in the same manner as described above (1-2) were cut with scissors, and the exudate from the cut surface was collected. The collected liquid was immediately diluted 10-fold with distilled water, and then 50 μl of the spore suspension (5 × 10 4The exudate was added to the plate at a final concentration of 1-0.001% (v / v) (cells / mL). After the assay plate was left in the dark at 25°C for 20-24 hours, the spores in the suspension were stained and fixed with lactophenol cotton blue solution, and the number of germinated and ungerminated spores (60-200 spores) observed in one field of view at a magnification of 150x under an optical microscope (OPTIPHOT-2, Nikon) was counted. The germination rate of spores was calculated as the average of three repeated counts.

[0050] (1-4) Induction of spore germination using homogenate from leaves and stems A leaf or about 3 cm of the stem of a sweet potato seedling grown in the same manner as described above (1-2) was cut off and crushed in a mortar until the tissue was fragmented. The liquid accumulated in the mortar was collected and 50 μl of the spore suspension (5 × 10 4 The homogenate was added to the culture medium (100 ml / mL) so that the final concentration of the homogenate was 1-0.001% (v / v). The conditions for the assay plate treatment and the observation method after treatment were the same as those described above in (1-3). The germination rate of spores was calculated from the results of one repeated observation.

[0051] (1-5) Induction of spore germination by sugars believed to be contained in the exudate As sugars likely to be contained in the exudate, we focused on D(+)-fucose, sucrose, glucose, fructose, galactose, rhamnose, and L(-)-fucose. Each sugar was dissolved in distilled water to prepare aqueous solutions of 10%, 1%, and 0.1% (w / v) (10 times the concentration at the time of testing). The aqueous solutions were prepared immediately before use. Using these aqueous solutions, 50 μl of spore suspension (5 × 10 4Spore suspensions containing no sugar and sweet potato stem exudate (1-3) at a final concentration of 1% were also prepared in wells as controls. After the assay plates were left in the dark at 25°C for 20-24 hours, the spores in each suspension were stained and fixed with lactophenol cotton blue solution, and germinated and ungerminated spores were counted under an optical microscope (BZ-X700, Keyence) for 90-110 spores per well. The germination rate of spores was calculated as the average of triplicate counts.

[0052] (2) Experimental results (2-1) Observation of spore germination on sweet potato tissue Alpha spores of the sweet potato root rot fungus germinated near wounds on the stems, petioles, and leaves of sweet potatoes, but hardly germinated in undamaged areas (Figure 1).

[0053] (2-2) Induction of spore germination by stem exudates Based on the findings above (2-1), we focused on the components of exudates such as yarapin that seep out from the wound site and investigated their spore germination-inducing activity. As a result, we found that the exudates induced spore germination of root rot fungi, and the degree of inducing activity was dependent on the concentration of the exudate (Figure 2).

[0054] (2-3) Induction of spore germination using homogenate from leaves and stems Not only stem exudates but also leaf and stem homogenates induced spore germination (Fig. 4).

[0055] (2-4) Induction of spore germination by sugars believed to be contained in the exudate Among the sugars tested, D(+)-fucose, one of the constituent sugars of yarapin, induced germination of α-spores (Fig. 5), whereas L(-)-fucose, the optical isomer of D(+)-fucose, did not induce germination (Fig. 6). [Industrial Applicability]

[0056] The present invention can be used in the agricultural field and the like.

Claims

1. A spore germination inducer for sweet potato root rot fungus, which contains D(+)-fucose or yarapin as an active ingredient.

2. A method for inducing spore germination of sweet potato root rot fungus, comprising a step of contacting the spore germination inducer of sweet potato root rot fungus according to claim 1 with spores of sweet potato root rot fungus.

3. A method for controlling sweet potato root rot, comprising the following steps (1) and (2): (1) A step of applying the spore germination inducer of sweet potato root rot fungus according to claim 1 to a control object to germinate spores of sweet potato root rot fungus present in the control object; (2) After step (1), a step of applying a control agent for sweet potato root rot to the control object.

4. 4. The method for controlling sweet potato root rot according to claim 3, wherein the object to be controlled originates from a field where a plant of the Convolvulaceae family is cultivated.

5. The method for controlling sweet potato root rot according to claim 3, characterized in that the object to be controlled is derived from a field where sweet potatoes are grown.

6. A method for screening for a substance that inhibits spore germination induction of sweet potato root rot fungus, comprising the following steps (1) and (2): (1) contacting the spore germination inducer of sweet potato root rot fungus according to claim 1 with spores of sweet potato root rot fungus in the presence of a test substance; (2) After the step (1), a step of observing the germination state of spores of the sweet potato root rot fungus.

7. A method for detecting sweet potato root rot fungus, comprising the following steps (1) and (2): (1) contacting a sample suspected of containing sweet potato root rot fungus with the spore germination inducer of sweet potato root rot fungus according to claim 1; (2) After step (1), a step of detecting sweet potato root rot fungus.

8. The method for detecting sweet potato root rot fungus according to claim 7, characterized in that the sample suspected of containing sweet potato root rot fungus is derived from a plant body of the Convolvulaceae family or from a field where a plant of the Convolvulaceae family is cultivated.

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