Method for subculturing obligate parasitic fungus using aseptically cultured plant body

Abstract

To provide a technique enabling subculturing of an obligate parasitic fungus.SOLUTION: The present invention provides a method for subculturing an obligate parasitic fungus using an aseptically cultured plant body.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for subculturing an obligate parasite using a sterile cultured plant.

Background Art

[0002] Among the bacteria that cause diseases in plants such as grapes, there are obligate parasites that can only grow on living plants. Examples of such obligate parasites include grape downy mildew bacteria. Obligate parasites, for example, invade through the stomata of plants and cannot be cultured on artificial media, and always require leaves of parasitic plants. For these reasons, research on obligate parasites has not progressed.

[0003] Patent Documents 1 and 2 describe subculturing obligate parasites such as grape downy mildew bacteria and cucumber powdery mildew bacteria using leaves of ordinary plants.

[0004] However, with ordinary plants, it takes time to increase the seedlings, and it is also difficult to maintain the plants without getting sick.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

[0006] The present inventors have found that an obligate parasite can be subcultured by using a sterile cultured plant. The present invention is based on this finding.

[0007] Therefore, the present invention provides a method for subculturing an obligate parasite by using a sterile cultured plant.

[0008] The present invention encompasses the following inventions. (1) A method for subculturing an obligate parasite, comprising the step of culturing the obligate parasite using a sterile cultured plant. (2) The method according to (1), wherein the obligate parasite is a bacterium that causes plant diseases in grapes and / or hops. (3) The method according to (1) or (2), wherein the obligate parasite is at least one selected from the group consisting of downy mildew bacteria, powdery mildew bacteria, and rust bacteria. (4) The method according to any one of (1) to (3), wherein the obligate parasite is at least one selected from the group consisting of grape downy mildew bacterium (Plasmopara viticola), hop downy mildew bacterium (Pseudoperonospora humuli), grape powdery mildew bacterium (Erysiphe necator Schweinitz var. necator), hop powdery mildew bacterium (Oidium sp., Podosphaera macularis), and grape rust bacterium (Phakopsora meliosmae-myrianthae). (5) The method according to any one of (1) to (4), wherein the sterile cultured plant is selected from grapes and hops. (6) The method according to any one of (1) to (5), wherein the sterile cultured plant contains at least one part selected from leaves and leaf stalks. (7) A step of preparing leaves of a sterile cultured plant, a step of inoculating a suspension containing conidia of the obligate parasite or conidia of the obligate parasite onto the leaves of the sterile cultured plant, and a step of culturing the obligate parasite inoculated onto the leaves of the sterile cultured plant The method according to any one of (1) to (6), comprising the above steps. (8) The method according to (7), wherein the leaf age of the leaves of the sterile cultured plant is 90 days or less. (9) The method according to (7) or (8), wherein the method for inoculating the suspension or conidia is selected from the group consisting of the micropipette dropping method, the spray spraying method, and the conidia dropping method. (10) A method for detecting the tolerance of an obligate parasite to a pesticide according to any one of (1) to (9), comprising: a step of applying a pesticide to a sterile cultured plant body in which an obligate parasite obtained by the method according to any one of (1) to (9) is subcultured; and a step of further culturing the obligate parasite and the sterile cultured plant body, and then confirming the onset of the obligate parasite A method for detecting tolerance to a pesticide, comprising the steps of: (11) The method according to (10), wherein the pesticide is at least one pesticide selected from the group consisting of a QoI fungicide, a thiazole carboxamide, an OSBPI fungicide, an SDHI fungicide, a CAA fungicide, cyanoacetamide-oxime, a PA fungicide, a chloronitrile, a QiI fungicide, a DMI fungicide, and a dithiocarbamate.

[0009] According to the present invention, a method for subculturing an obligate parasite is provided. The present invention is advantageous in that the subcultured obligate parasite can be constantly provided as a test sample in various tests. The present invention is further advantageous in that the obligate parasite can be subcultured aseptically.

[0010] Furthermore, in the prevention of plant diseases such as grapes, control with pesticides is common, but the emergence of pesticide-resistant bacteria is known. In order to carry out effective pesticide control, it is necessary to correctly grasp the emergence status of resistant bacteria in the field and select appropriate pesticides. In the determination of resistant bacteria, the leaf disk method and the PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method using non-sterile plant bodies are known. Here, examples of non-sterile plant bodies include potted seedlings and field plant bodies.

[0011] The present invention is further advantageous in detecting the resistance of obligate parasites to pesticides. For example, by using aseptic cultured plants, the present invention does not require a cultivation space for preparing non-aseptic plants, has no limited seasons for preparing non-aseptic plants like this, and is also advantageous in ensuring that the plants are not infected with non-target fungi. In addition, although the PCR-RFLP method can only evaluate limited pesticides because gene mutation sites involved in drug resistance need to be identified, the present invention is advantageous in that the pesticides that can be evaluated are not limited and no equipment for performing PCR is required by detecting the resistance of obligate parasites to pesticides using aseptic cultured plants.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

[0013] One feature of the method for subculturing the obligate parasite of the present invention is that it includes a step of culturing the obligate parasite using an aseptic cultured plant.

[0014] Obligate parasite The absolute parasitic fungi used in the present invention are not particularly limited as long as they can obtain nutrients only from living cells of the host and cannot be cultured on synthetic media. According to one embodiment of the present invention, such absolute parasitic fungi are preferably fungi that cause plant diseases in grapes and / or hops.

[0015] According to another embodiment of the present invention, examples of the above absolute parasitic fungi include downy mildew, powdery mildew, and anthracnose fungi or combinations thereof, and preferably downy mildew and powdery mildew.

[0016] According to a preferred embodiment of the present invention, examples of the above absolute parasitic fungi include grape downy mildew (Plasmopara viticola), hop downy mildew (Pseudoperonospora humuli), grape powdery mildew (Erysiphe necator Schweinitz var. necator), hop powdery mildew (Oidium sp., Podosphaera macularis), and grape anthracnose (Phakopsora meliosmae-myrianthae) or combinations thereof, and preferably grape downy mildew, hop downy mildew, grape powdery mildew, and hop powdery mildew.

[0017] Sterile cultured plant The aseptically cultured plants used in the present invention are not particularly limited as long as they are aseptically cultured, and examples include grapes and hops, and preferably grapes and hops. Furthermore, examples of grape varieties include Syrah, Merlot, 3309, Gravesac, and 5BB, and preferably Syrah and Merlot, and more preferably Syrah. Furthermore, examples of hop varieties include Saaz and Hallertauer, and preferably Saaz. It should be noted that the growth rates of grapes and hops under aseptic culture are similar.

[0018] Examples of the part of the aseptically cultured plant used in the present invention include leaves and leaf stalks, and leaves are preferably used.

[0019] As the method for producing the aseptically cultured plant, a known method such as growing and / or propagating the plant in a culture system under aseptic conditions after subjecting the plant to aseptic treatment can be used. When the aseptically cultured plant is grape, methods such as heat treatment method and shoot tip culture method can be used as the method for producing the aseptically cultured plant. As the shoot tip culture method, for example, according to the method described in 'Hiroyuki Sasahara et al., "Fruit Tree Grape", 1990, pp. 253-255, The World of Plant Tissue Culture (supervised by Harumitsu Higuchi), Shibata Hario Glass Co., Ltd.', an aseptically cultured plant can be produced. When the aseptically cultured plant is hop, methods such as heat treatment method and shoot tip culture method can be used as the method for producing the aseptically cultured plant. As the shoot tip culture method, for example, according to the method described in 'Hiroyuki Sasahara et al., "Fruit Tree Grape", 1990, pp. 253-255, The World of Plant Tissue Culture (supervised by Harumitsu Higuchi), Shibata Hario Glass Co., Ltd.', an aseptically cultured plant can be produced.

[0020] Method for subculturing obligate parasite using sterile cultured plant According to one embodiment of the present invention, a method for subculturing an obligate parasite using the aseptically cultured plant of the present invention includes a step of preparing an aseptically cultured plant, a step of inoculating a suspension containing conidia of the obligate parasite or conidia of the obligate parasite onto the aseptically cultured plant, and a step of culturing the aseptically cultured plant after inoculation. Aseptic culture technology is a technology that utilizes the totipotency of plant differentiation to aseptically regenerate and / or maintain plant individuals from plant nodes. By using aseptically cultured plants, it is advantageous in providing a culture method for obligate parasites that saves space, has rapid individual growth, can prepare plants without seasonal restrictions, and ensures asepticity.

[0021] According to a preferred embodiment of the present invention, a method for subculturing an obligate parasite using the aseptically cultured plant body of the present invention includes the steps of preparing a leaf of the aseptically cultured plant body, inoculating a suspension containing conidia of the obligate parasite or conidia of the obligate parasite onto the leaf of the aseptically cultured plant body, and culturing the leaf of the aseptically cultured plant body after inoculation.

[0022] <Step of preparing a leaf of the aseptically cultured plant body> The step of preparing a leaf of the aseptically cultured plant body is not particularly limited, but includes the steps of cutting a node of a stem containing a growth point of the aseptically cultured plant body produced by aseptic culture, inserting the node into a medium and culturing it, and obtaining a leaf of the aseptically cultured plant body obtained by the culture. As the above plant, grape and hop are preferable, and grape is more preferable.

[0023] As the aseptically cultured plant body used in the step of cutting a node of a stem containing a growth point of the aseptically cultured plant body produced by the above aseptic culture, it is preferable that the plant body has 3 or more leaves and has rooted. The above cutting step is preferably carried out aseptically using a clean bench or the like.

[0024] Specifically, in the step of inserting the above node into the medium and culturing it, it is preferable to insert the node of the stem containing the growth point into the medium with the root side facing down. The number of nodes per plant box is 1 to 4 nodes, preferably 2 to 3 nodes. The medium used in the culture is not particularly limited, but is preferably a 1 / 2MS medium containing no hormones.

[0025] In the step of obtaining leaves of the aseptically cultured plant body obtained by the above culture, the leaf age of the leaves, that is, the leaf age of the leaves to which the obligate parasite is inoculated, is preferably 90 days or less. From the viewpoint of being able to surely perform subculture, it is more preferably 40 to 90 days, still more preferably 60 to 80 days, and still more preferably 60 to 79 days. Here, the leaf age is the age of the leaf when the day when the node of the stem including the growing point is inserted into the medium is set as day 0. The leaves to which the obligate parasite is inoculated are more preferably expanded leaves. For example, aseptically cultured grapes at a leaf age of around 60 to 70 days usually have about 6 leaves per aseptically cultured grape plant body, of which 1 or less are immature leaves and the rest are expanded leaves. Here, an expanded leaf is a leaf in which the leaf margin at the base of the leaf blade is expanded, while an immature leaf is a leaf in which the leaf margin at the base of the leaf blade is not expanded, and such a leaf may further have the characteristics of being lighter in color or significantly smaller. Also, stomata are formed in expanded leaves, but not in immature leaves. An example of an expanded leaf and an immature leaf is shown in FIG. 1.

[0026] <Step of inoculating a suspension containing conidia of the obligate parasite onto the leaves of the aseptically cultured plant body> As a method for preparing a suspension containing conidia of the obligate parasite in the step of inoculating a suspension containing conidia of the obligate parasite onto the leaves of the aseptically cultured plant body, a method of obtaining a conidial suspension of the obligate parasite from a diseased leaf of the obligate parasite by watering the diseased part of the obligate parasite in the diseased leaf of the obligate parasite (specifically, the conidiophore and the conidia formation site) and suspending the conidia in water on the leaf can be mentioned. Further, it is preferable to repeat the above method at a plurality of diseased parts and mix the obtained suspensions. Examples of the amount of the above water include 20 to 100 μL, preferably 40 to 80 μL, and it can be quantified using a micropipette or the like.

[0027] The concentration of conidia in the conidial suspension of the obligate parasite inoculated onto the leaves of the aseptically cultured plant body is, for example, 5×10 3 ~1×10 6 cells / mL, and from the viewpoint of easier success of infection, it is preferably 1×10 4 ~5×10 5It is set to / mL.

[0028] The leaf used in the above inoculation step is a leaf cut off from the aseptically cultured plant body at the petiole part. When inoculating, it is preferably placed on wet filter paper or the like with the inoculation surface (preferably the abaxial side or the adaxial side of the leaf) facing up.

[0029] According to one embodiment of the present invention, although not bound by theory, for filamentous fungi belonging to the family Erysiphaceae, such as powdery mildew fungi, since the hyphae invade from the stomata on the abaxial side of the leaf, it is preferable to inoculate the abaxial side of the leaf for filamentous fungi belonging to the family Erysiphaceae, such as grape powdery mildew. According to another embodiment of the present invention, although not bound by theory, for fungi of the order Erysiphales, such as grape powdery mildew and hop powdery mildew, since it is known that the hyphae grow on the adaxial side of the leaf, it is preferable to inoculate the adaxial side of the leaf for fungi of the order Erysiphales, such as grape powdery mildew and hop powdery mildew.

[0030] In the step of inoculating a suspension containing conidia of the obligate parasite obtained from the diseased leaves of the obligate parasite onto the leaves of the aseptically cultured plant, the method of inoculating the suspension containing the conidia is not particularly limited, but examples include the micropipette dropping method and the spray spraying method, and preferably the micropipette dropping method. As the micropipette dropping method, a micropipette with a dispensing volume of 10 to 100 μL is used, and water droplets are evenly dropped onto the underside or the entire surface of the leaf. The volume of the water droplets is, for example, 2 to 10 μL, and from the viewpoint of the infection success rate, it is preferably 3 to 6 μL. For example, 20 to 70 water droplets can be dropped per leaf, and preferably 40 to 60 water droplets are dropped. Also, when dropping, the tip of the micropipette tip may slightly touch the leaf, but it is preferable not to scratch the epidermis. As the spray spraying method, the conidia suspension is filled into a spray, and the entire leaves of the aseptically cultured plant are uniformly spray-inoculated. The amount of the conidia suspension sprayed by the spray can be appropriately set according to the type and size of the leaves of the plant to be sprayed. For example, 100 to 1000 μL can be mentioned, and preferably 200 to 500 μL.

[0031] <Step of inoculating conidia of the obligate parasite onto the leaves of the aseptically cultured plant> In the step of inoculating conidia of the obligate parasite onto the leaves of the aseptically cultured plant, the method of inoculating conidia of the obligate parasite includes the conidia brushing-off method. As the conidia brushing-off method, conidia are taken from the lesion of the diseased leaf using a painting brush or the like, and the conidia are gently applied with a painting brush or the like to the entire underside or surface of the aseptically cultured leaf so as not to damage the underside or surface of the leaf for smear inoculation. The number of conidia of the obligate parasite inoculated onto the leaves of the aseptically cultured plant is not particularly limited, but for example, 10 to 100 conidia per 1 mm 2 of leaf area can be mentioned. The leaves and the inoculation surface of the conidia used in the step of inoculating conidia of the obligate parasite are the same as those in the <Step of inoculating a suspension containing conidia of the obligate parasite onto the leaves of the aseptically cultured plant>.

[0032] According to a preferred embodiment of the combination of an obligate parasite and its inoculation method, the obligate parasite is Plasmopara viticola, and its inoculation method is the micropipette dropping method or the spray spraying method, more preferably the micropipette dropping method. According to another preferred embodiment of the combination of an obligate parasite and its inoculation method, the obligate parasite is Uncinula necator, and its inoculation method is the micropipette dropping method, the spray spraying method and the conidium shaking-off method, more preferably the micropipette dropping method. According to another preferred embodiment of the combination of an obligate parasite and its inoculation method, the obligate parasite is Erysiphe humuli, and its inoculation method is the micropipette dropping method, the spray spraying method and the conidium shaking-off method, more preferably the conidium shaking-off method. According to another preferred embodiment of the combination of an obligate parasite and its inoculation method, the obligate parasite is Pseudoperonospora humuli, and its inoculation method is the micropipette dropping method, the spray spraying method and the conidium shaking-off method, more preferably the micropipette dropping method.

[0033] <Step of culturing an obligate parasite inoculated on the leaf of a sterile cultured plant> The step of culturing an obligate parasite (preferably Plasmopara viticola, Uncinula necator, Erysiphe humuli) inoculated on the leaf of a sterile cultured plant is not particularly limited, but it is preferably carried out under high humidity. Examples of high humidity include a state where the filter paper in a petri dish is sufficiently moistened with water, specifically, a state where the filter paper in a 9 cm petri dish is moistened with 5 to 9 mL of water or a humidity equivalent to that state. Also, when culturing at a position close to the air outlet in the growth chamber, it is preferable to seal a part of the outer periphery (for example, about 70% of the outer periphery) with masking tape to maintain the humidity in the petri dish.

[0034] As culture conditions for the obligate parasite (preferably, Plasmopara viticola, Uncinula necator, Uncinula necator of hops), there are no particular limitations as long as the obligate parasite can be cultured, and those skilled in the art can arbitrarily select and set them depending on the type of obligate parasite, the concentration of the conidia suspension, the number of conidia, etc. Examples of the culture temperature include 15 to 30°C, preferably 20 to 25°C. Examples of the culture period include 7 to 13 days, preferably 8 to 12 days. Examples of the light-dark conditions include 14 to 18 hours of light irradiation / 6 to 10 hours of dark conditions, preferably 15 to 17 hours of light irradiation / 7 to 9 hours of dark conditions.

[0035] According to a preferred embodiment of the present invention, if observation is performed 7 days or more after inoculation with the obligate parasite (preferably, Plasmopara viticola, Uncinula necator, Uncinula necator of hops) and sufficient conidia generation is observed, in the procedure of the above-described steps of inoculating the suspension containing conidia or conidia to the aseptic cultured plant body (preferably, grape, hop) and culturing the aseptic cultured plant body after inoculation, by inoculating the obligate parasite to the leaves of the new aseptic cultured plant body, the obligate parasite can be subcultured and maintained.

[0036] According to a preferred embodiment of the present invention, by including the step of culturing the obligate parasite using an aseptic cultured plant body, the obligate parasite can be subcultured. The number of conidia of the obligate parasite obtained by the step of culturing the obligate parasite using an aseptic cultured plant body is, for example, the number of conidia per 1 mm 2 of leaf area. From the viewpoint of enabling subculture more easily, the number of conidia is 1 or more, preferably 1 to 1000, and from the viewpoint of more stable subculture, more preferably 10 to 100. The number of conidia per 1 mm 2 of the above leaf area can be calculated as follows. First, after suspending and collecting all the conidia of the diseased leaf with water, a part is injected into a cell counter plate and measured with an optical microscope to calculate the conidia concentration [number / mL] in the conidia suspension. Next, the conidia concentration [number / mL] is multiplied by the volume [mL] of the suspension to calculate the number of conidia [number] per leaf. The number of conidia [number] per leaf is divided by the leaf area (mm2 ) divided by the leaf area of 1 mm 2 , the number of conidia per unit area can be calculated.

[0037] According to another preferred embodiment of the present invention, as the infection efficiency of the obligate parasite obtained by culturing the obligate parasite using an aseptic cultured plant, from the viewpoint of enabling subculture, for example, a disease index of 2 or more can be mentioned, preferably the disease index is 2 to 4, and from the viewpoint of more stable subculture, more preferably the disease index is 3 to 4. Here, the disease index is as follows. 0: No disease spots are observed on the leaves, 1: The disease spot area is less than 5% of the leaf area, 2: The same is 5% or more and less than 25%, 3: The same is 25% or more and less than 50%, 4: The same is 50% or more. For example, when culturing using a plurality of leaves of an aseptic cultured plant, at least one leaf only needs to show the above disease index.

[0038] According to one embodiment of the present invention, regarding the number of conidia of the obligate parasite (preferably, grape downy mildew pathogen, grape powdery mildew pathogen) that can be recovered from one aseptic leaf (preferably, aseptic cultured grape leaf) based on the above disease index, the following relationship can be mentioned as a guideline. · When the disease index is 3 - 4, the number of conidia that can be recovered from one leaf is 1×10 4 or more and 1×10 5 or less, and stable subculture of the obligate parasite (preferably, grape downy mildew pathogen, grape powdery mildew pathogen) is possible. · When the disease index is 2, the number of conidia that can be recovered from one leaf is 1×10 3 or more and less than 1×10 4 , and subculture of the obligate parasite (preferably, grape downy mildew pathogen, grape powdery mildew pathogen) is possible. · When the disease index is 1, the number of conidia that can be recovered from one leaf is 1×10 2 or more and less than 1×10 3 , and subculture of the obligate parasite (preferably, grape downy mildew pathogen, grape powdery mildew pathogen) is impossible.

[0039] According to another embodiment of the present invention, regarding the number of conidia of an obligate parasite (preferably, Plasmopara viticola, Uncinula necator) that can be recovered from one aseptic leaf (preferably, an aseptic cultured grape leaf) based on the above disease index, the following relationships can be cited as a guide. · When the disease index is 3 - 4, the number of conidia that can be recovered from one leaf is 3×10 3 or more and 1×10 5 or less, and stable subculture of the obligate parasite (preferably, Plasmopara viticola, Uncinula necator) is possible. · When the disease index is 2, the number of conidia that can be recovered from one leaf is 5×10 2 or more and 3×10 3 less than, and subculture of the obligate parasite (preferably, Plasmopara viticola, Uncinula necator) is possible. · When the disease index is 1, the number of conidia that can be recovered from one leaf is 1×10 2 or more and 5×10 2 less than, and subculture of the obligate parasite (preferably, Plasmopara viticola, Uncinula necator) is impossible.

[0040] Method for detecting pesticide tolerance in obligate parasite According to another aspect of the present invention, there is provided a method for detecting the resistance of an obligate parasite to a pesticide (also referred to as drug resistance), comprising the steps of applying a pesticide to an aseptic cultured plant body in which the obligate parasite has been subcultured, and after further culturing the obligate parasite and the aseptic cultured plant body, confirming the onset of the obligate parasite. The method for detecting the resistance of the present invention to a pesticide is advantageous in that it can detect the resistance of the obligate parasite. Further, the method for detecting the resistance of the present invention to a pesticide is advantageous in that it enables the verification of the effect of a pesticide (also referred to as a control agent) on the obligate parasite and / or the acceleration of the detection of resistance to a pesticide. Also, it is advantageous in that it can detect the resistance to a pesticide regardless of the specific situation of the gene mutation site.

[0041] <Step of applying (spraying) a pesticide to an aseptic cultured plant body in which the obligate parasite has been subcultured> As the aseptic cultured plant body used in the step of applying a pesticide to the aseptic cultured plant body in which the above-described obligate parasite is subcultured, an aseptic cultured plant body obtained by the method of subculturing the above-described obligate parasite is preferable.

[0042] According to one embodiment of the present invention, the method of applying a pesticide to an aseptic cultured plant body in which the above-described obligate parasite is subcultured is not particularly limited, and examples thereof include spraying (for example, foliar spraying), dipping, and coating, and preferably, spraying.

[0043] According to a preferred embodiment of the present invention, the timing of applying a pesticide to the above-described aseptic cultured plant body is not particularly limited, and examples thereof include 0 to 4 days after inoculation with the fungus, preferably 1 to 3 days after inoculation with the fungus, and more preferably 2 days after inoculation with the fungus.

[0044] <Step of further culturing the obligate parasite and the aseptic cultured plant body and then confirming the onset of the obligate parasite> According to one embodiment of the present invention, the method for detecting the tolerance of the obligate parasite of the present invention to a pesticide preferably includes the step of further culturing the obligate parasite and the aseptic cultured plant body after applying the pesticide to the aseptic cultured plant body as described above, and the step of then confirming the onset of the above-described obligate parasite.

[0045] According to one embodiment of the present invention, when culturing the obligate parasite and the aseptic cultured plant body after applying a pesticide to the aseptic cultured plant body, the culturing period is not particularly limited. For example, it may be 5 to 12 days after the pesticide application, preferably 7 to 10 days, and more preferably 8 to 9 days. According to another embodiment of the present invention, when culturing the obligate parasite and the aseptic cultured plant body after applying a pesticide to the aseptic cultured plant body, the culturing period is not particularly limited. For example, it may be 7 to 14 days after the inoculation of the fungus, preferably 9 to 12 days, and more preferably 10 to 11 days. In addition, the culturing conditions in the further culturing after the pesticide application are the same as the culturing conditions before the pesticide application, that is, the culturing conditions in the step of culturing the obligate parasite inoculated on the leaves of the aseptic cultured plant body described above.

[0046] According to a preferred embodiment of the present invention, the method for confirming the onset of the obligate parasite is not particularly limited. For example, the above-mentioned disease index of the obligate parasite can be used. When using the disease index, the onset can be, for example, a weak onset when the disease index is 1 or more and less than 2, a moderate onset when it is 2 or more and less than 3, and a strong onset when it is 3 or more.

[0047] <Pesticide> The pesticide used in the present invention is not particularly limited as long as it has an effect on the obligate parasite and can cause drug resistance. For example, as the group name in the FRAC code, quinone outside inhibitor (QoI) fungicides, thiazole carboxamide, OSBPI agents, SDHI fungicides, CAA fungicides, cyanoacetamide = oxime, PA fungicides, chloronitrile, QiI fungicides, DMI fungicides and dithiocarbamate can be mentioned. Preferably, QoI fungicides, QiI fungicides, DMI fungicides, PA fungicides, and dithiocarbamate are used, and more preferably QoI fungicides are used.

[0048] Examples of QoI fungicides include, for example, methoxyacrylates, methoxyacetamides, methoxycarbamates, oxime acetates, oxime acetamides and dihydrodioxazine fungicides, as well as oxazolidinediones, imidazolinones and benzylcarbamates, tetrazolinone fungicides. Preferably, they are methoxyacrylates, oxazolidinediones, benzylcarbamates. Examples of methoxyacrylates include azoxystrobin, kresoxim-methyl, enoxastrobin (also known as enestroburin), fluoxastrobin, picoxystrobin and pyraoxystrobin. Examples of methoxyacetamides include mandestrobin. Examples of methoxycarbamates include pyraclostrobin, pyrametostrobin and triclopyricarb. Examples of oxime acetates include kresoxim-methyl and trifloxystrobin. Examples of oxime acetamides include dimoxystrobin, fenaminstrobin, metominostrobin and orysastrobin. Examples of dihydrodioxazines include fluoxastrobin. Examples of oxazolidinediones include famoxadone. Examples of imidazolinones include fenamidone. Examples of benzylcarbamates include pyribencarb.

[0049] Examples of thiazole carboxamides include ethylaminothiazole carboxamides such as ethaboxam, and preferably ethaboxam.

[0050] Examples of oxysterol-binding protein inhibitors (OSBPIs) fungicides include piperidinylthiazole isoxazolines such as oxathiapiprolin and fluoxapiprolin, and preferably oxathiapiprolin.

[0051] Examples of succinate dehydrogenase inhibitor (SDHI) fungicides include phenylbenzamides, phenyloxoethylthiophenamides, pyridinylethylbenzamides, thiazole carboxamides, pyrazole-4-carboxamides, N-methoxyphenylethylpyrazole carboxamides, pyridine carboxamides, and pyrazine carboxamides, preferably pyrazole-4-carboxamides. Examples of phenylbenzamides include flutolanil and mepronil. Examples of phenyloxoethylthiophenamides include isofetamid. Examples of pyridinylethylbenzamides include fluopyram. Examples of thiazole carboxamides include difenoconazole. Examples of pyrazole-4-carboxamides include fluxapyroxad, flutianil, ipfencarbazone, isopyrazam, penthiopyrad, and sedaxane. Examples of N-methoxyphenylethylpyrazole carboxamides include pydiflumetofen. Examples of pyridine carboxamides include boscalid. Examples of pyrazine carboxamides include pyraclostrobin.

[0052] Examples of carboxylic acid amide (CAA) fungicides include cinnamic acid amides, valinamide carbamates, and mandelic acid amide fungicides. Examples of cinnamic acid amides include dimethomorph, flumorph, and pyrimorph. Examples of valinamide carbamates include benthiavalicarb, benthiavalicarb-isopropyl, iprovalicarb, tolprocarb, and valifenalate (also known as valifenal). Examples of mandelic acid amides include mandipropamid, N-[2-[4-[[3-(4-chlorophenyl)-2-propyn-1-yl]oxy]-3-methoxyphenyl]ethyl]-3-methyl-2-[(methylsulfonyl)amino]butanamide, and N-[2-[4-[[3-(4-chlorophenyl)-2-propyn-1-yl]oxy]-3-methoxyphenyl]ethyl]-3-methyl-2-[(ethylsulfonyl)amino]butanamide, preferably mandipropamid.

[0053] Examples of cyanoacetamide-oxime include cymoxanil.

[0054] Examples of phenylamide (PA) fungicides include acylalanine, oxazolidinone, and butyrolactone fungicides, preferably acylalanine. Examples of acylalanine include benalaxyl, benalaxyl-M (also known as kiralaxyl), furalaxyl, metalaxyl, and metalaxyl-M (also known as mefenoxam). Examples of oxazolidinone include oxadixyl. Examples of butyrolactone include ofurace.

[0055] Examples of chloronitrile include aromatic rings substituted with chloro and cyano, specifically chlorothalonil (TPN).

[0056] Examples of quinone-inside inhibitor (QiI) fungicides include cyanoimidazole and sulfamoyl triazole. Examples of cyanoimidazole include cyazofamid. Examples of sulfamoyl triazole include amisulbrom.

[0057] Examples of demethylation reaction inhibitors (DMIs) used as fungicides include piperazine, pyrimidine, imidazole, triazole, and triazolylthione. Preferred examples are imidazole and triazole. Examples of piperazine include triforine. Examples of pyrimidine include fenarimol. Examples of imidazole include oxpoconazole fumarate, pefurazoate, prochloraz, and triflumizole. Examples of triazole include cyproconazole, difenoconazole, fenbuconazole, hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil, propiconazole,simeconazole, tebuconazole, tetraconazole, triticonazole, and mefentrifluconazole. An example of triazolylthione is prothioconazole.

[0058] Examples of dithiocarbamates include manzeb, mancozeb, propineb, thiram, and ziram. Preferred examples are manzeb and thiram.

[0059] According to a preferred embodiment of the combination of obligate parasites and pesticides, the obligate parasite is a downy mildew pathogen, preferably Plasmopara viticola, the pesticide is a fungicide, preferably a QoI fungicide, more preferably azoxystrobin.

[0060] The pesticide can be used in any available form or formulation (also referred to as a pesticide formulation). Examples of such formulations include liquid formulations such as wettable powders, aqueous suspensions, dispersions, emulsions, powders, water-soluble formulations, and flowables. Preferred are wettable powders, liquid formulations, powders, and flowables. The content of the pesticide (also referred to as the pesticide component) in the above formulations can be appropriately adjusted by those skilled in the art according to the type of pesticide, etc. Here, in the formulation, the above pesticide may be used alone or in combination of two or more. When used in combination of two or more, the content of the pesticide in the above formulation refers to the total content of two or more pesticides.

[0061] The pesticide formulation can be used, for example, as a spray, a coating agent, or a dipping agent. When diluting the prepared pesticide formulation to produce an aqueous composition (also referred to as a dilution) containing the pesticide formulation, water can be used. The dilution ratio when preparing the aqueous composition can be appropriately selected according to the plants, obligate parasites, etc. to be used, and can be, for example, 50 to 5000 times, preferably 500 to 3000 times.

[0062] When using the pesticide formulation by spraying, depending on the type and content of the pesticide, generally an aqueous composition of the pesticide formulation diluted with water about 100 to 5000 times can be sprayed. Also, the application rate, application time, and application method of the pesticide formulation and the aqueous composition containing the pesticide formulation can be appropriately determined according to the pesticide components to be formulated.

Examples

[0063] The present invention will be specifically described based on the following examples, but the present invention is not limited to these examples.

[0064] Measurement of conidia number For the number of conidia of the grape downy mildew pathogen in each test example, after all the conidia on the diseased leaves (that is, the aseptically cultured grape leaves infected with the grape downy mildew pathogen) were suspended and collected with water, a part was injected into a cell counter plate (177 - 512C, manufactured by WATSON), and measured with an optical microscope (magnification 100 times).

[0065] Disease index The infection results can also be evaluated by the disease index. The specific evaluation criteria for the disease index are shown. 0: No disease spots are observed on the leaves 1: The diseased spot area is less than 5% of the leaf area 2: The diseased spot area is 5% or more and less than 25% of the leaf area 3: The diseased spot area is 25% or more and less than 50% of the leaf area 4: The diseased spot area is 50% or more of the leaf area

[0066] Test Example 1 (Examination of leaf age of sterile cultured grape leaves) Test Plot 1-1 Culture of sterile cultured plant (sterile cultured grape leaves) Sterile cultured grapes (varieties: Syrah, Merlot, 3309, Gravesac, 5BB) were produced by the "shoot tip culture method" described in "Hiroyuki Sasahara et al., 'Fruit Tree Grape', 1990, pp. 253-255, The World of Plant Tissue Culture (supervised by Haruzo Higuchi), Shibata Hario Glass Co., Ltd.". The sterile cultured grapes used were rooted plants with 4 to 6 leaves. As the culture medium for the sterile cultured grapes, a hormone-free 1 / 2 MS medium (containing 3% sucrose and 0.8% agar, pH 5.8) was dispensed into plant culture boxes (made of polycarbonate, inner dimensions 65.4 mm × 65.4 mm × 98.2 mm) at about 50 mL each and sterilized ones were used. The water accumulated in the plant culture box was drained immediately before use. Under aseptic conditions in a clean bench (BCB-3E7, ESCO), the nodes of the stem containing the growing point were cut with a scalpel blade, and 2 nodes per plant culture box were inserted into the medium with the root side facing down, and cultured in a growth chamber (CLE-405, Tommy Seiko Co., Ltd.) at 25°C under a 16-hour light irradiation / 8-hour dark condition.

[0067] Culture of obligate parasite (grape downy mildew pathogen) On the day of inoculation with Plasmopara viticola, the expanded leaves of sterile cultured grapes with a leaf age of 68 or 70 days were cut off from the petiole with a scalpel blade and immediately placed on a 9-cm plastic petri dish with the leaf back facing up. Two sheets of sterilized filter paper were placed in the petri dish, and the filter paper was moistened by dispensing 7 mL of sterilized water.

[0068] 50 μL of sterilized water was applied to the diseased parts (i.e., conidiophores and conidium formation sites) of diseased grape leaves collected in the field in advance with a micropipette, suspended on the leaf, and a conidium suspension was prepared. The same operation was repeated for each diseased part, a large amount of the suspension was collected, and pipetting was performed to uniformly disperse the suspension. The concentration of conidia in the suspension was 1×10 4 ~1×10 5It was cells / mL. Inoculation was carried out by evenly dropping approximately 50 droplets of 3 - 5 μL using a 20 μL or 50 μL micropipette onto the entire underside of the leaf (hereinafter also referred to as the micropipette dropping method). When dropping, the tip of the micropipette tip may slightly touch the leaf, but it was carefully performed so as not to damage the epidermis by pinching. After inoculation, the lid of the petri dish was closed, and cultivation was started in a growth chamber set at 22 °C under 16 - hour light irradiation / 8 - hour dark conditions. When culturing at a position close to the air outlet in the growth chamber, about 70% of the outer circumference was sealed with masking tape (720N - 18, Nitto Denko Corporation) to maintain the humidity inside the petri dish. Cultivation was carried out for 7 - 13 days.

[0069] The number of conidia per 1 mm 2 of leaf area obtained in Test Plot 1 - 1 on the 8th or 9th day after inoculation with grape downy mildew pathogen (i.e., after 8 or 9 days of cultivation after inoculation with grape downy mildew pathogen) was calculated. The results using the Shira variety as aseptic - cultured grapes are shown in Table 1.

Table 1

[0070] Furthermore, for the leaves at 70 - day leaf age in Test Plot 1 - 1, the disease index was such that the number of leaves with disease index 0 was 0, the number of leaves with disease index 1 was 0, the number of leaves with disease index 2 was 0, the number of leaves with disease index 3 was 0, and the number of leaves with disease index 4 was 4.

[0071] Test Plot 1-2 In Test Plot 1 - 2, aseptic - cultured grapes and grape downy mildew pathogen were cultured in the same manner as in Test Plot 1 - 1, except that leaves at 47 - day leaf age were used as the expanded leaves of aseptic - cultured grapes in the "cultivation of grape downy mildew pathogen" of Test Plot 1 - 1.

[0072] The number of conidia per 1 mm 2 of leaf area obtained in Test Plot 1 - 2 on the 9th day after inoculation with grape downy mildew pathogen was calculated. The results using the Shira variety as aseptic - cultured grapes are shown in Table 2.

Table 2

[0073] Test Plot 1-3 In Test Plots 1-3, for the expanded leaves of aseptically cultured grapes in the "Cultivation of Grape Downy Mildew Pathogen" of Test Plot 1-1, those with a leaf age of 80 days were used, and the measurement of the number of conidia was carried out 7 days after inoculation with the grape downy mildew pathogen. For aseptically cultured grapes with a leaf age of 80 days, after cutting with a scalpel blade, the leaves quickly wither and the infection efficiency decreases. Therefore, when using aseptically cultured grapes in the 80-day leaf age range, after placing them in the above-mentioned petri dishes, they were left standing in a growth chamber (22°C, 16-hour light irradiation / 8-hour dark condition) until the next day, and an operation to exclude the leaves with tea-colored leaf margins was added. Except for the above, the aseptically cultured grapes and the grape downy mildew pathogen were cultured in the same manner as in Test Plot 1-1.

[0074] The number of conidia per 1 mm 2 of leaf area at 80 days after inoculation with the grape downy mildew pathogen obtained in Test Plot 1-3 was calculated. The results using the Shira variety as the aseptically cultured grapes are shown in Table 3.

Table 3

[0075] Test Plot 1-4 In Test Plot 1-4, for the expanded leaves of aseptically cultured grapes in the "Cultivation of Grape Downy Mildew Pathogen" of Test Plot 1-1, those with a leaf age of 82 days were used, and the evaluation by the disease index was carried out 8 days after inoculation with the grape downy mildew pathogen. Regarding the aseptically cultured grapes with a leaf age of 82 days, the exclusion operation as in Test Plot 1-3 was not performed. Except for the above, the aseptically cultured grapes and the grape downy mildew pathogen were cultured in the same manner as in Test Plot 1-1.

[0076] The disease index of the leaves with a leaf age of 82 days in Test Plot 1-4 was such that the number of leaves with a disease index of 0 was 0, the number of leaves with a disease index of 1 was 4, the number of leaves with a disease index of 2 was 1, the number of leaves with a disease index of 3 was 0, and the number of leaves with a disease index of 4 was 0.

[0077] Test Plot 1-5 In Test Sections 1-5, leaves at 88 days old of aseptically cultured grapes, which were fully expanded leaves, were used. Except for performing the exclusion operations as in Test Sections 1-3, aseptically cultured grapes and the cultivation of grape downy mildew pathogen were carried out in the same way as in Test Section 1-4.

[0078] The disease incidence index of the leaves at 88 days old in Test Section 1-5 was as follows: 0 leaves had a disease incidence index of 0, 0 leaves had an index of 1, 0 leaves had an index of 2, 2 leaves had an index of 3, and 2 leaves had an index of 4.

[0079] From the results of Test Sections 1-4 and 1-5, by adding the operation of excluding the leaves with tea-colored leaf margins, an infection efficiency comparable to that when using leaves at 60 - 70 days old as fully expanded leaves of aseptically cultured grapes was observed.

[0080] Test Plot 1-6 In Test Section 1-6, except for using leaves at 93 days old as fully expanded leaves of aseptically cultured grapes in the "cultivation of grape downy mildew pathogen" of Test Section 1-1, aseptically cultured grapes and the cultivation of grape downy mildew pathogen were carried out in the same way as in Test Section 1-1.

[0081] In Test Section 1-6, the number of conidia per 1 mm 2 of leaf area was calculated 8 days after inoculation with grape downy mildew pathogen. The results using the Shira variety as aseptically cultured grapes are shown in Table 4.

Table 4

[0082] Test Plot 1-7 In Test Section 1-7, except for using leaves at 103 days old as fully expanded leaves of aseptically cultured grape plants in the "cultivation of grape downy mildew pathogen" of Test Section 1-1 and performing the evaluation by disease incidence index 8 days after inoculation with grape downy mildew pathogen, aseptically cultured grapes and the cultivation of grape downy mildew pathogen were carried out in the same way as in Test Section 1-1.

[0083] In Test Plot 1-7, for the leaves at leaf age 103 days, there were 3 leaves with disease index 1 and 2 leaves with disease index 2.

[0084] Test Example 2 (Difference in infection due to difference in inoculation method) Test Plot 2-1 (Micro pipette dropping method) In Test Plot 2-1, leaves at leaf age 70 days were used as the expanded leaves of aseptic-cultured grapes in the "Cultivation of Grape Downy Mildew Pathogen" of Test Plot 1-1. The measurement of the number of conidia was carried out 7 days after inoculation with Grape Downy Mildew Pathogen. The aseptic-cultured grapes and the cultivation of Grape Downy Mildew Pathogen were carried out in the same manner as in Test Plot 1-1, except that the following conidia suspension was used as the suspension. The conidia suspension was obtained by repeatedly pipetting 50 μL of sterile water onto the lesions of the diseased leaves of Grape Downy Mildew Pathogen and suspending the conidia on the leaves. The concentration of conidia in the suspension was 4×10 4 ~5×10 4 individuals / mL. Note that the leaves at leaf age 70 days in Test Plot 2-1 are different leaves from those in Test Plot 1-1. The results are shown in Table 5 and Figure 2A. Note that the lower middle leaf in Figure 2A was curled, so it was not included in the test data.

[0085] Test Plot 2-2 (Spray method) In Test Plot 2-2, the aseptic-cultured grapes and the cultivation of Grape Downy Mildew Pathogen were carried out in the same manner as in Test Plot 2-1, except that the inoculation was performed by the spray method. Here, the spray method was carried out as follows. 50 μL of sterile water was pipetted onto the lesions of the diseased leaves of Grape Downy Mildew Pathogen, and the conidia were suspended on the leaves. The same operation was repeated to prepare a conidia suspension. The concentration of conidia in the suspension was 4×10 4 ~5×10 4 individuals / mL. The obtained conidia suspension was filled into a spray (made of plastic, 2 mL capacity) and uniformly spray-inoculated onto the entire aseptic-cultured grape leaves (spray volume: 300 - 400 μL). The results are shown in Table 5 and Figure 2B.

[0086] Test Plot 2-3 (Conidia brushing-off method) In Test Plot 2-3, aseptic culture of grapes and grape downy mildew was carried out in the same manner as in Test Plot 2-1, except that inoculation was performed by the method of brushing off conidia. Here, the method of brushing off conidia was carried out as follows. Conidia were taken from the lesions of grape downy mildew-infected leaves using a paintbrush, and the conidia were gently applied with the paintbrush to the entire abaxial surface of the aseptic culture grape leaves so as not to damage the abaxial surface for inoculation by painting. The results are shown in Table 5 and Figure 2C.

Table 5

[0087] The disease index of the cultured leaves in Test Plot 2-1 was such that the number of leaves with disease index 1 was 0, the number of leaves with disease index 2 was 0, the number of leaves with disease index 3 was 2, and the number of leaves with disease index 4 was 1. The disease index of the cultured leaves in Test Plot 2-2 was such that the number of leaves with disease index 1 was 1, the number of leaves with disease index 2 was 2, the number of leaves with disease index 3 was 1, and the number of leaves with disease index 4 was 0. Also, the disease index of the cultured leaves in Test Plot 2-3 was such that the number of leaves with disease index 0 was 4, the number of leaves with disease index 1 was 0, the number of leaves with disease index 2 was 0, the number of leaves with disease index 3 was 0, and the number of leaves with disease index 4 was 0.

[0088] The number of conidia that could be recovered from one aseptic culture leaf with successful infection in Test Plot 2-1 (micro pipette dropping method) was 6.0×10 4 individuals, 1.1×10 4 individuals, 2.4×10 4 individuals. The number of conidia that could be recovered from one aseptic culture leaf with successful infection in Test Plot 2-2 (spraying method) was 7.5×10 2 individuals, 3.1×10 3 individuals, 2.7×10 3 individuals, 4.5×10 2 individuals. The number of conidia that could be recovered from one aseptic culture leaf with successful infection in Test Plot 2-3 (method of brushing off conidia) was 0 for all (4 leaves).

[0089] Test Example 3 (Examination of grape powdery mildew pathogen) Test Plot 3-1 Culture of sterile cultured plant (sterile cultured grape leaves) Aseptic culture grapes were obtained by the same culture method as "cultivation of grape downy mildew" in Test Plot 1-1.

[0090] Culture of obligate parasite (grape powdery mildew pathogen) On the day of inoculation with Erysiphe necator Schweinitz var. necator, the expanded leaves of aseptically cultured grapevines at a leaf age of 87 days were cut off from the petiole with a scalpel blade and immediately placed on a 9-cm plastic Petri dish with the leaf surface facing up. Two sheets of sterilized filter paper were placed in the Petri dish, and 7 mL of sterilized water was dispensed to moisten the filter paper. In aseptically cultured grapevines at a leaf age of 87 days, the leaves rapidly wither and the infection efficiency decreases after cutting with a scalpel blade. Therefore, when using aseptically cultured grapevines in their 80s in terms of leaf age, after placing them in the above Petri dish, they were left standing in a growth chamber (22 °C, 16-hour light irradiation / 8-hour dark condition) until the next day, and an operation to exclude the leaves with browned leaf margins was added.

[0091] 50 μL of sterilized water was applied to the diseased parts (i.e., conidiophores and conidia-forming parts) of diseased grapevine leaves collected in the field in advance with a micropipette, suspended on the leaf, and a conidia suspension was prepared. The same operation was repeated for each diseased part, a large amount of the suspension was collected, and pipetting was performed to uniformly disperse the suspension. The concentration of conidia in the suspension was 1×10 4 ~1×10 5 cells / mL. For inoculation, approximately 50 drops of 3 - 5 μL droplets were evenly dropped onto the entire leaf surface using a 20 μL or 50 μL micropipette (hereinafter also referred to as the micropipette dropping method). When dropping, the tip of the micropipette tip may slightly touch the leaf, but it was carefully performed so as not to scratch the epidermis. After inoculation, the lid of the Petri dish was closed, and culturing was started in a growth chamber set at 22 °C, 16-hour light irradiation / 8-hour dark condition. When culturing at a position close to the air outlet in the growth chamber, about 70% of the outer periphery was sealed with masking tape (720N - 18, Nitto Denko Corporation) to maintain the humidity inside the Petri dish. The culturing was carried out for 7 - 13 days. Evaluation based on the disease index was performed 13 days after inoculation with Erysiphe necator Schweinitz var. necator.

[0092] In Test Plot 3-1, for the leaves at the 87-day leaf age, the disease incidence index was as follows: 0 diseased leaves had an index of 0, 1 diseased leaf had an index of 1, 1 diseased leaf had an index of 2, 3 diseased leaves had an index of 3, and 0 diseased leaves had an index of 4.

[0093] Test Plot 3-2 (Conidia brushing-off method) In Test Plot 3-2, aseptic culture of grapes and grape powdery mildew fungus was carried out in the same manner as in Test Plot 3-1, except that the inoculation was performed by the method of brushing off conidia. Here, the method of brushing off conidia was as follows. Conidia were taken from the lesion of a grape powdery mildew-infected leaf using a paintbrush, and the conidia were gently spread over the entire leaf surface of the aseptic culture grape leaves with a paintbrush so as not to damage the leaf surface, and smear inoculation was performed.

[0094] In Test Plot 3-2, for the leaves at the 83-day leaf age, the disease incidence index was as follows: 0 diseased leaves had an index of 0, 1 diseased leaf had an index of 1, 1 diseased leaf had an index of 2, 2 diseased leaves had an index of 3, and 0 diseased leaves had an index of 4.

[0095] Test Example 4 (Examination of sterile cultured hop leaves) Test Plot 4-1 Culture of sterile cultured plant (sterile cultured hop leaves) Aseptic culture hops (variety: Saaz) were produced by the "shoot tip culture method" described in "Hiroyuki Sasahara et al., 'Fruit Tree - Grape', 1990, pp. 253 - 255, The World of Plant Tissue Culture (supervised by Haruzo Higuchi), Shibata Hario Glass Co., Ltd.". As the culture medium for aseptic culture grapes, a hormone-free 1 / 2MS medium (containing 2% glucose and 0.8% agar, pH 5.8) was dispensed into plant culture boxes (made of polycarbonate, inner dimensions 65.4 mm × 65.4 mm × 98.2 mm) at about 50 mL each and sterilized for use. The water accumulated in the plant culture box was drained immediately before use. Under aseptic conditions in a clean bench (BCB-3E7, ESCO), the nodes of the stem containing the growing point were cut with a scalpel blade, and 2 nodes per plant culture box were inserted into the medium with the root side facing down, and cultured in a growth chamber (CLE-405, Tommy Seiko Co., Ltd.) at 20°C under a 16-hour light irradiation / 8-hour dark condition.

[0096] Culture of obligate parasite (hop powdery mildew pathogen) On the day of inoculation with powdery mildew fungi (Oidium sp., Podosphaera macularis), leaves of aseptically cultured hops at 84 days old were cut off from the petiole with a scalpel and immediately placed on a 9-cm plastic Petri dish with the leaf surface facing up. Two sheets of sterilized filter paper were laid in the Petri dish, and 7 mL of sterilized water was dispensed to moisten the filter paper. In aseptically cultured hops at 84 days old, after cutting with a scalpel, the leaves rapidly wilt and the infection efficiency decreases. Therefore, when using aseptically cultured hops in the 80-day-old stage, after placing them in the above Petri dish, they were left standing in a growth chamber (22 °C, 16-hour light irradiation / 8-hour dark condition) until the next day, and an operation to exclude leaves with tea-colored leaf margins was added.

[0097] In Test Plot 4-1, aseptically cultured hop leaves were used as aseptically cultured plants, and powdery mildew fungi were used as obligate parasites. The aseptically cultured hops and powdery mildew fungi were cultured in the same manner as in Test Plot 3-1, except that the inoculation method was the conidium brushing-off method. Here, the conidium brushing-off method was performed as follows. Conidia were taken from the lesion of the diseased leaf using a paintbrush, and the conidia were gently painted with the paintbrush on the entire leaf surface of the aseptically cultured hop leaves so as not to damage the leaf surface for smear inoculation.

[0098] In Test Plot 4-1, for the leaves at 84 days old, the disease index was as follows: 11 leaves had a disease index of 0, 0 leaves had a disease index of 1, 0 leaves had a disease index of 2, 0 leaves had a disease index of 3, and 1 leaf had a disease index of 4.

[0099] Test Plot 4-2 Culture of sterile cultured plant (sterile cultured hop leaves) and obligate parasite (hop powdery mildew pathogen) Aseptically cultured hop leaves and powdery mildew fungi were cultured using aseptically cultured hops (cultivar: Health Brucker). In Test Plot 4-2, the aseptically cultured hops and powdery mildew fungi were cultured in the same manner as in Test Plot 4-1, except that the cultivar of hops used was Health Brucker. As a result, in Test Plot 4-2, for the leaves at 84 days old, the disease index was as follows: 14 leaves had a disease index of 0, 0 leaves had a disease index of 1, 0 leaves had a disease index of 2, 0 leaves had a disease index of 3, and 1 leaf had a disease index of 4.

[0100] Test Example 5 (Detection of drug-resistant bacteria) Test Plots 5-1 to 5-5 Culture of sterile cultured plant (sterile cultured grape leaves) Sterile grapes were obtained by using the same culture method as "cultivation of grape downy mildew pathogen" in Test Plot 1-1.

[0101] Culture of obligate parasite (grape downy mildew pathogen) The grape downy mildew pathogen was cultured in the same manner as in "cultivation of grape downy mildew pathogen" in Test Plot 1-1. Specifically, the grape downy mildew pathogen was cultured by the following method. On the day of inoculation of the grape downy mildew pathogen, the expanded leaves of sterile grapes with a leaf age of 68 days were cut off from the petiole with a scalpel blade, and three expanded leaves were quickly placed on a 9-cm plastic Petri dish with the leaf back facing up. Two sheets of sterilized filter paper were laid in the Petri dish, and the filter paper was moistened by dispensing 5 mL of sterilized water.

[0102] 50 μL of sterilized water was applied to the diseased parts (i.e., conidiophores and conidium formation sites) of grape downy mildew-diseased leaves collected in the field in advance with a micropipette, suspended on the leaf, and a conidium suspension was prepared. The same operation was repeated for each diseased part, a large amount of the suspension was collected, and pipetting was performed to uniformly disperse the suspension. The concentration of conidia in the suspension was 5×10 4 ~1×10 5 cells / mL. Inoculation was carried out by dropping approximately 50 droplets of 3 - 6 μL evenly over the entire leaf back using a 20-μL or 50-μL micropipette. When dropping, the tip of the micropipette tip may slightly touch the leaf, but it was carefully performed so as not to scratch the epidermis. After inoculation, the lid of the Petri dish was closed, and cultivation was started in a growth chamber set at 20 - 25°C under 16-hour light irradiation / 8-hour dark conditions. When culturing at a position close to the air outlet in the growth chamber, about 70% of the outer periphery was sealed with masking tape (720N-18, Nitto Denko Corporation) to maintain the humidity in the Petri dish. Cultivation was carried out for 10 days.

[0103] Spraying of pesticide Test Plots 5-1 to 5-5 In Test Plots 5-1 to 5-5, the pesticides were used as pesticide formulations and applied at the manufacturer's recommended concentrations. Specifically, in Test Plot 5-1, first, azoxystrobin (10.0%) wettable powder (Amistar 10 Flowable, manufactured by Syngenta Japan Co., Ltd.) as a pesticide formulation was diluted 1000-fold with sterilized water to obtain a diluted solution. Two days after inoculating grape downy mildew pathogen, the diluted solution was spray-sprayed onto the leaves so that it was 500 - 600 μL per petri dish (i.e., per 3 expanded leaves). Then, cultivation was carried out, and evaluation was performed based on the disease index 8 days after pesticide spraying (i.e., 10 days after inoculating grape downy mildew pathogen). In Comparative Plot 5-1, it was carried out in the same manner as Test Plot 5-1 except that only sterilized water was used without using pesticides. The results are shown in Table 6.

[0104]

Table 6

[0105] In Test Plot 5-2, it was carried out in the same manner as Test Plot 5-1 except that ethaboxam wettable powder (Ethiphen Flowable, manufactured by Nippon Soda Co., Ltd.) containing 12.5% ethaboxam was used as a pesticide formulation. In Test Plot 5-3, it was carried out in the same manner as Test Plot 5-1 except that oxathiapiprolin (2.7%) · mandipropamid (23.0%) wettable powder (Orondis Ultra SC, manufactured by Syngenta Japan Co., Ltd.) was used as a pesticide formulation and the dilution multiple was 2000-fold. In Test Plot 5-4, it was carried out in the same manner as Test Plot 5-1 except that cymoxanil (30.0%) · famoxadone (22.5%) wettable powder (Horizon Dry Flowable, manufactured by Nissan Chemical Industries, Ltd.) was used as a pesticide formulation and the dilution multiple was 2500-fold. In Test Plot 5-5, it was carried out in the same manner as Test Plot 5-1 except that metalaxyl-M (3.3%) · TPN (32.0%) wettable powder (Folio Gold, manufactured by Syngenta Japan Co., Ltd.) was used as a pesticide formulation and the dilution multiple was 1500-fold. The results are shown in Table 6.

[0106] As a result of the evaluation based on the disease incidence index, in test plot 5-1, all three leaves had a disease incidence index of 4. Also, in comparison plot 5-1, all three leaves had a disease incidence index of 4. In test plots 5-2 to 5-5, all three leaves had a disease incidence index of 0 respectively. Therefore, only in test plot 5-1 where the QoI fungicide was used, the disease incidence index was equivalent to that of the sterilized water treatment plot which was comparison plot 5-1. From this, it was suggested that the fungus used in the test was a QoI fungicide-resistant bacterium.

[0107] Test Example 6 (Detection of drug-resistant bacteria) According to the method described in Furuya, S., Suzuki, S., Kobayashi, H., Saito, S., & Takayanagi, T. (2009). Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations. Pest Management Science: formerly Pesticide Science, 65(8), 840-843., the gene diagnosis of the grape downy mildew fungus used in Test Example 5 above was carried out by the nested PCR-RFLP method. The results are shown in Figure 3. The right lane in Figure 3A is the band shown by the grape downy mildew fungus in Test Example 5. Here, it is known that there are two bands for the QoI fungicide-resistant bacterium. On the other hand, the bands of the QoI fungicide-sensitive bacterium are shown in the right two lanes of Figure 3B. The QoI fungicide-sensitive bacterium shows only one band. Therefore, it was found that the grape downy mildew fungus used in Test Example 5 was a QoI fungicide-resistant bacterium. Therefore, it was confirmed that equivalent results could be obtained even with the drug resistance detection method using the method of subculturing obligate parasites in axenic cultured plants.

Claims

1. A method for subculturing an obligate parasite, comprising the step of culturing the obligate parasite using a sterile cultured plant.

2. The method according to claim 1, wherein the obligate parasite is a bacterium that causes plant diseases in grapes and / or hops.

3. The method according to claim 1 or 2, wherein the obligate parasite is at least one selected from the group consisting of downy mildew bacteria, powdery mildew bacteria, and rust bacteria.

4. The method according to claim 3, wherein the obligate parasite is at least one selected from the group consisting of grape downy mildew (Plasmopara viticola), hop downy mildew (Pseudoperonospora humuli), grape powdery mildew (Erysiphe necator Schweinitz var. necator), hop powdery mildew (Oidium sp., Podosphaera macularis), and grape rust (Phakopsora meliosmae-myrianthae).

5. The method according to claim 1 or 2, wherein the sterile cultured plant is selected from grapes and hops.

6. The method according to claim 1 or 2, wherein the sterile cultured plant comprises at least one part selected from leaves and leaf stalks.

7. A step of preparing leaves of a sterile cultured plant, A step of inoculating a suspension containing conidia of the obligate parasite or conidia of the obligate parasite onto the leaves of the sterile cultured plant, and A step of culturing the obligate parasite inoculated onto the leaves of the sterile cultured plant The method according to claim 1 or 2, comprising:

8. The method according to claim 7, wherein the leaf age of the leaves of the sterile cultured plant is 90 days or less.

9. The method according to claim 8, wherein the inoculation method of the suspension or conidia is selected from the group consisting of the micropipette dropping method, the spray spraying method, and the conidia brushing-off method.

10. A method for detecting the tolerance of the obligate parasite according to claim 1 to pesticides, comprising: A step of applying a pesticide to a sterile cultured plant in which the obligate parasite obtained by the method according to claim 1 is subcultured, and After further culturing the obligate parasite and the axenically cultured plant, a step of confirming the onset of the obligate parasite A method for detecting resistance to pesticides, comprising:

11. The method according to claim 10, wherein the pesticide is at least one pesticide selected from the group consisting of a QoI fungicide, a thiazole carboxamide, an OSBPI fungicide, an SDHI fungicide, a CAA fungicide, cyanoacetamide = oxime, a PA fungicide, a chloronitrile, a QiI fungicide, a DMI fungicide, and a dithiocarbamate.

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