JP6713117B1 - Plant pathogen control agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a compound having an excellent controlling effect against phytopathogenic fungi, and to provide a controlling agent and a controlling method for the phytopathogenic fungus using the compound. It has been found that it has an excellent controlling effect. [Selection diagram] None

Description

The present invention relates to a phytopathogen control agent containing a metal chelate or a salt as an active ingredient, and a phytopathogen control method comprising applying the control agent to a plant.

Humans rely on plants for much of their food and useful substance production. Plant diseases such as filamentous fungal diseases, bacterial diseases, and plant viral diseases are one of the major factors that significantly reduce the productivity of plants, and if cultivated without protection against plant diseases, the yield would be 70%. It is expected that the revenue will decrease. Thus, the reduction of crop yield loss by controlling plant diseases has an effect comparable to the increase in production by mass cultivation of crops.

Therefore, various control agents have been developed for protecting crops from plant diseases. For example, against plant diseases caused by infection with phytopathogenic fungi such as filamentous fungal disease and bacterial disease, control agents mainly containing antibiotics kasugamycin and streptomycin, strobilurin fungicides (QoI agents), succinic acid dehydrogenation Control agents such as enzyme inhibitors (SDHI agents) are used. However, it has been pointed out that these control agents have the problem that resistant bacteria develop in a few years, making disease control difficult.

On the other hand, as a control agent for plant diseases caused by plant virus infection, although lentemin has been registered as a pesticide in Japan, a chemical pesticide that is a silver bullet does not yet exist (lentemin is a registered trademark of Noda Shokuhin Kogyo Co., Ltd.). Agricultural Chemical Registration No. 15584, No. 17774, No. 19439, No. 19440).

Under such circumstances, the present inventor has found that metal chelates have a controlling effect against plant viruses, and proposes to utilize them as a controlling agent against plant viruses (Patent Document 1).

JP 2017-178838

The present invention has been made in view of such circumstances, and an object thereof is to identify a compound having an excellent control effect against plant pathogens, and a control agent for the plant pathogens using the compound and a control method. To provide.

As a result of intensive studies to solve the above problems, the present inventor has found that various metal chelates or salts have excellent control effects against phytopathogenic fungi.

It has been reported that metal chelates have a controlling effect against plant viruses (Patent Document 1). However, since the plant virus and the plant pathogen have completely different mechanisms of infecting the plant, it is not possible to predict whether a drug that exerts a control effect on the plant virus will also exert a control effect on the plant pathogen. This is supported by the fact that lentemin, which is the only pesticide against plant viral diseases, did not suppress filamentous fungal diseases at all (see Reference Example 1). Therefore, it is surprising that metal chelates and salts exerted a controlling effect not only on plant viruses but also on plant pathogens.

In addition, the control effect against phytopathogenic fungi by metal chelate and salt was very high, and it was recognized against a wide range of phytopathogenic fungi that infects Arabidopsis thaliana, Pakchoi, tomato, cucumber, strawberry and the like. Furthermore, when a metal chelate was used, no phytotoxicity to plants occurred even when administered at high concentrations.

From the above, the present inventor has found that metal chelates and salts are effective as control agents against phytopathogenic fungi, and particularly for metal chelates, they can be control agents having both excellent control effect and safety, The present invention has been completed.

The present invention relates to a control agent for phytopathogenic fungi containing a metal chelate or salt as an active ingredient, and a method for controlling phytopathogenic fungi, which comprises applying the control agent to plants, and more specifically, to provide the following inventions. Is.

[1] A plant pathogen control agent containing a metal chelate or salt as an active ingredient.

[2] The control agent according to [1], wherein the metal is copper or zinc.

[3] The control agent according to [1], wherein the metal chelate is prepared using gluconic acid as a chelating agent.

[4] The control agent according to [1], wherein the metal chelate is selected from the group consisting of copper gluconate, zinc gluconate, and zinc glycine.

[5] The control agent according to any of [1] to [4], wherein the plant pathogen is a plant pathogen or a plant pathogen.

[6] A method for controlling phytopathogenic fungi, which comprises applying the control agent according to any one of [1] to [4] to plants.

[7] The control method according to [6], wherein the plant pathogen is a plant pathogen or a plant pathogen.

By using the control agent of the present invention, it is possible to effectively control phytopathogenic fungi. In particular, a control agent containing a metal chelate as an active ingredient does not cause phytotoxicity such as leaf atrophy and spotting, and is therefore excellent in safety. Further, according to the control agent of the present invention, it is possible to enhance the expression of the defense response gene of the plant and induce the resistance of the plant to the phytopathogenic bacterium, and the risk of producing a resistant bacterium is small. Furthermore, in the plant body, it is possible to bring about a controlling effect not only in the part to which it is applied but also in the peripheral part.

It is a graph which shows the result of having analyzed the expression of the defense response gene in Arabidopsis treated with various metal chelates. In the figure, CuGluc indicates copper gluconate, ZnGluc indicates zinc gluconate, and FeGluc indicates iron gluconate. The same applies below. It is a graph which shows the infection inhibitory effect of the Brassicaceae plant black spot bacterial pathogen in the bok choy which was treated with zinc gluconate. As the varieties, Qing Tei and Xiao Pao were used. It is a graph which shows the infection inhibitory effect of the Brassicaceae plant black spot bacterial pathogen in the bok choy which various metal chelate process was carried out. Shaopao was used as the variety. In the figure, CuSO ₄ indicates copper sulfate, and ZnSO ₄ indicates zinc sulfate. The same applies below. It is a graph which shows the infection inhibitory effect of the Brassicaceae vegetable anthracnose fungus in the bok choy which various metal chelate process was carried out. Shaopao was used as the variety. It is a graph which shows the infection inhibitory effect of the Brassicaceae vegetable anthracnose fungus in Arabidopsis treated with various metal chelates. It is a graph which shows the infection inhibitory effect of the Brassicaceae plant black spot bacterial pathogen in Arabidopsis treated with various metal chelates. It is a graph which shows the infection inhibitory effect of the leaf bacillus disease fungus in tomatoes which were treated with various metal chelates. It is a graph which shows the infection inhibitory effect of the cucumber anthracnose fungus in the cucumber which various metal chelate processing was carried out. It is a graph which shows the infection inhibitory effect of Strawberry anthracnose in strawberry processed with various metal chelates.

The metal chelate or salt used in the control agent of the present invention is not particularly limited as long as it has an effect of controlling plant pathogens.

Examples of the metal used in the present invention include, for example, copper, zinc, iron, manganese, molybdenum, magnesium, silicon, boron, calcium, cobalt, nickel, etc., but since they have low toxicity to plants, copper or zinc is preferable. Is.

When the metal ion is applied to the soil as it is (inorganic form), the metal ion may combine with phosphoric acid in the soil to form an insoluble precipitate, and the desired effect may not be sufficiently obtained. Therefore, a chelate is prepared in advance by combining with a chelating agent, and by applying the chelate, the combination with other components can be prevented and the original effect can be exhibited. In addition, metal chelates have the property of being easily absorbed by plants. From such a viewpoint, a chelate of a metal is preferable as an active ingredient of the control agent of the present invention.

In the present invention, a chelate compound or chelate salt is generally called a chelate.

Examples of the chelating agent for preparing a metal chelate include gluconic acid, glycine, EDTA (Ethylenediaminetetraacetic acid), DTPA (Diethylenetriamine pentaacetic acid), NTA (Nitrilotriacetic acid, Nitrilotriacetic acid). ), EDDS (Ethylenediamine-N,N'-disuccinic acid, Ethylenediamine-N,N'-disuccinic acid), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ), citric acid, formic acid, phytic acid, ethylenediamine, bipyridine, phenanthroline, etc., but gluconic acid or glycine is preferable because of its low toxicity to plants. Since copper gluconate, zinc gluconate, and zinc glycine have both excellent control effects and low toxicity, they can be most preferably used as active ingredients of the control agent of the present invention.

From the viewpoint of versatility, an EDTA-based chelating agent can be preferably used, and from the viewpoint of spontaneous decomposition, EDDS and the like can be preferably used.

Examples of metal salts include sulfates, phosphates, nitrates, chlorides and carbonates, but are not particularly limited as long as they are metal salts.

In the control agent of the present invention, the above active ingredients can be used alone or in combination of two or more kinds.

Further, the control agent of the present invention may contain any other component as long as the control effect of phytopathogenic bacteria is not impaired. Other optional components include, for example, fillers, extenders, binders, moisturizers, disintegrants, lubricants, diluents, excipients, surfactants, spreading agents, amino acids, peptides, fertilizers. Elements, ingredients derived from natural products and the like can be mentioned.

The control agent of the present invention can be used in combination with a known agent that contributes to the control of phytopathogenic fungi, in order to further enhance the control effect on phytopathogenic fungi or to expand the range of phytopathogenic fungi to be applied. It can also be used in combination with known insecticides, acaricides, plant virus control agents and the like.

The method for applying the control agent of the present invention to plants is not particularly limited, and examples thereof include spraying, coating, soil irrigation, soil admixture, application in nursery boxes, and seed disinfection. Since a very high infection control effect is observed in the area where the control agent of the present invention is applied, the application method includes application to the entire plant or leaves by spraying or application, or soil irrigation or soil admixture. It is preferable to apply it to the root by the method such as. The control agent of the present invention is advantageous in that it can exhibit a control effect against phytopathogenic bacteria even in the peripheral area of the applied area.

The dosage form of the control agent of the present invention can take various forms depending on the application method and the like. For example, emulsions, oils, aerosols, liquid agents such as flowable agents, wettable powders, water solvents, and powders. , Granules and the like. When applied to plants by spraying or the like, a dosage form that can be liquefied at the time of application is preferable.

The concentration of each drug at the time of application is not particularly limited as long as it has a controlling effect against phytopathogenic fungi and does not cause phytotoxicity to plants. Usually within the range of 0.1 to 50 mM, for example, if zinc gluconate, 2 to 20 mM is preferable, if iron gluconate, 0.2 to 2 mM is preferable, if copper gluconate, 0.1 to 5 mM, With glycine zinc, 2-20 mM is preferred. Moreover, 0.1-5 mM is preferable for zinc sulfate, and 0.1-5 mM is preferable for copper sulfate.

The timing of application of the control agent of the present invention to plants is not particularly limited, but preventive control is effective.

The control action of phytopathogenic bacteria in the control agent of the present invention includes an action of suppressing/inhibiting infection of phytopathogenic bacteria, an action of suppressing/inhibiting growth/extension/migration, and an action of killing. The phytopathogenic fungi on which the controlling agent of the present invention exerts a controlling action is not particularly limited, and may be bacteria or fungi such as filamentous fungi.

Examples of phytopathogenic bacteria include Pseudomonas spp, Erwinia spp, Xanthomonas spp, Ralstonia spp, Streptomyces spp, Clavibacter spp, Agrobacterium spp, Curtobacterium spp, Acidovorax spp, Burkholderia spp. , Not limited to these.

Specific plant pathogenic bacteria include, for example, Pseudomonas syringae pv. maculicola, Pseudomonas cannabina pv. alisalensis, Pseudomonas syringae pv. tomato, and lettuce spoilage fungus (Pseudomonas). cichorii, Pseudomonas marginalis, Pseudomonas viridiflava), Pseudomonas viridiflava, Xanthomonas arboricola pv. pruni, Pseudomonas syringae pv. syringae, Brenneria nigrifluens, Erwinia nigrifluens, etc. (Pseudomonas syringae pv. syringae), soybean spotted bacterial disease fungus (Pseudomonas savastanoi pv. glycinea), cucumber spotted bacterial disease fungus (Pseudomonas syringae pv. lachrymans), tobacco field fungus (Pseudomonas syringae pv. tabse bacterium) syringae pv. pisi), Kiwifruit canker (Pseudomonas syringae pv. actinidiae), Chinese cabbage, cabbage, Japanese radish, lettuce and other soft-rot fungi (Erwinia carotovora), rice white leaf blight (Xanthomonas oryzae pv. oryzae), soybean leaf burn fungus Xanthomonas campestris pv. glycinea, Xanthomonas axonopodis pv. glycinea), cabbage and broccoli, etc., Brassicaceae black rot (Xanthomonas campestris pv. campestris), lettuce spot bacterial fungus (Xanthomonas axonopodis pv. anthracis bacterium) citri subsp. citri), tomato, eggplant, pepper, strawberry, ginger wilt (Ralstonia solanacearum), potato scab (Streptomyces spp.), tomato canker (Clavibacter michiganensis subsp. michiganensis), Asteraceae, Rosaceae plant Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium rhizogenes, Grape root rot, Agrobacterium vitis, Rhizobium radiobacter, Kidney bean wilt bacterial disease, Curtobacterium flaccumfaciens pv. flaccumfaciens, Tulip Disease (Curtobacterium flaccumfaciens pv. oortii), watermelon fruit blotch bacterial disease fungus (Acidovorax avenae subsp. citrulli), rice brown streak bacterium/corn brown streak bacterium (Acidovorax avenae subsp. avenae), rice blast fungus (Burkholderia glumae), Examples thereof include, but are not limited to, Burkholderia plantarii of rice, and Pectobacterium carotovorum, Pectobacterium atrosepticum.

Further, as a plant pathogenic fungus, for example, Colletotrichum spp, Phytophthora spp, Podosphaera spp, Sphaerotheca spp, Leveillula spp, Oidium spp, Oidiopsis spp, Erysiphe spp, Uncinula spp, Botrytis spp, Fusarium spp, Pyricularia spp, Fulvia spp, Pseudocercospora spp, Gibberella spp, Monographella spp, Pestalotiopsis spp, Corynespora spp, Puccinia spp, Alternaria spp, Plasmopara spp, Bremia spp, Peronospora spp Bacteria, Cochliobolus spp., Rhizoctonia spp., Sclerotinia spp., Verticillium spp., Venturia spp., Monilinia spp., Cercospora spp., Leptosphaeria spp. are not limited to these.

Specific plant pathogenic fungi include, for example, Brassicaceae anthracnose fungus (Colletotrichum higginsianum), Cucurbitaceae anthracnose fungus (Colletotrichum orbiculare), strawberry anthrax fungus (Colletotorichum acutatum, Colletotrichum gloeosporioides species complex; C. aenigma, C. aenigma. fructicola, C. siamense), anthracnose anthracnose (Colletotrichum incanum, Colletotrichum dematium), late rot of grape (Colletotrichum gloeosporioides), anthracnose anthracnose (Colletotrichum graminicola), anthracnose sp. ), potato and tomato blight (Phytophthora infestans), strawberry blight (Phytophthora nicotianae, Phytophthora cactorum, Phytophthora sp.), taro blight (Phytophthora colocasiae), vegetables, ornamental plants, tobacco, and other host plant blight. Phytophthora spp.), strawberry powdery mildew (Podosphaera aphanis, Sphaerotheca aphanis, Sphaerotheca humuli), tomato powdery mildew (Leveillula taurica, Oidium sp., Oidium lycopersici, Oidium neolycolyci, powdery mildew). Oidiopsis sicula, Erysiphe polygoni, Oidium sp.), barley and wheat powdery mildew (Erysiphe graminis), grape powdery mildew (Erysiphe necator, Uncinula necator), pea powdery mildew (Erysiphe pisi), squash powdery mildew , Oidium cit rulli), powdery mildew of eggplant (Erysiphe cichoracearum, Sphaerotheca fuliginea, Oidiopsis sicula), powdery mildew of vegetables, ornamental plants, other host plants, gray of tomatoes, strawberries, cucumbers, vegetables, grapes, other host plants Botrytis cinerea, Fusarium oxysporum f.sp. fragariae), rice blast fungus (Pyricularia grisea (P. oryzae)), tomato leaf mold fungus (Fulvia fulva), tomato scab (Pseudocercospora fuligena), wheat leaf mold (Gibberella zeae, Fusarium avenaceum, Fuwellium crusarum, Fusarium crumorum) , Monographella nivalis), tea leaf spot fungus (Pestalotiopsis longiseta, Pestalotiopsis theae), soybean acute withdrawal fungus (Fusarium tucumaniae, Fusarium virguliforme), cucumber brown spot fungus (Corynespora cassiicola), barley and wheat black rust graminis , Barley and wheat stripe rust (Puccinia striiformis Westendorp var. striiformis), barley small rust fungus (Puccinia hordei Otth), wheat red rust fungus (Puccinia recondita Roberge ex Desmazieres), green onion rust fungus (Puccinia allium) White rust fungus (Puccinia horiana Hennings), coffee, western pears, apples, peanuts, vegetables, ornamental plants, other host plant rust fungus, pear black spot fungus (Alternaria alternata), cabbage black rust fungus (Alternaria brassicicola), Chinese cabbage leaf spot fungus (Alternaria brassicae, Alternaria brassicicola, Alternaria japonica), other vegetables (for example, cucumber and cruciferous vegetables), apples, tomatoes, and other host plant leaf spot fungus (Alternaria spp.), grape downy mildew (Plasmopara) viticola), lettuce downy mildew (Bremia lactucae), cucumber downy mildew (Pseudoperonospora cubensis), Chinese cabbage downy mildew (Peronospora parasitica) ), soybean, tobacco, onion, other host plant downy mildew (Peronospora spp.), rice sesame leaf blight (Cochliobolus miyabeanus), cucumber vine (Fusarium oxysporum f. sp. cucumerinum), tomato wilt fungus (Fusarium oxysporum f. sp. lycopersici), rice bacillus seedling fungus (Gibberella fujikuroi), cucumber, eggplant and other Rhizoctonia solani, tomato small grain bacterium (Sclerotinia minor), potato half-body wilt fungus (Verticillium) albo-atrum, Verticillium dahliae, Verticillium nigrescens, Verticillium tricorpus), tomato ring fungus (Alternaria solani), vegetable sclerotinia fungus (Sclerotinia sclerotiorum), apple scab (Venturia inaequalis), peach aphidosis fungus (Monilinia fructicola) Examples include, but are not limited to, soybean purpura fungus (Cercospora kikuchii), sugar beet brown spot fungus (Cercospora beticola), and wheat wilt fungus (Leptosphaeria nodorum).

Representative examples of plants for controlling phytopathogenic fungi include, for example, tomato, eggplant, peppers, capsicum, tobacco, solanaceous plants such as potatoes, cucumber, pumpkin, melons and other cucurbitaceae plants, strawberries, apples, pears. , Rumeaceae plants such as plums, plums, rapeseed plants such as Chinese cabbage, bok choy, cabbage, komatsuna, Arabidopsis thaliana, legume plants such as soybean, rice plants such as rice and wheat, and taro plants such as taro. However, the present invention can be widely applied to many other plants infected with the above plant pathogens.

For the relationship between phytopathogenic fungi and host plants, refer to the Japanese Plant Disease Name Database (Agricultural Biological Resource Genebank).

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited to the following examples.

[Example 1] Analysis of expression of defense response gene in Arabidopsis treated with various metal chelates Arabidopsis thaliana (ecological type: Colombia) was used as a plant material, and soil (Supermix A (Sakata seed)): white vermiculite (A-2, (Asahi Kogyo Co., Ltd.): Obsidian perlite (Fuyolite No. 3)=2:1:1) and seeds were cultivated at 22° C. for a 24-hour light-dark cycle (light time 12 hours and dark time 12 hours). One week after sowing, the plants were replanted so that there were 5 individuals per pot, and the plants were further cultivated for 3 weeks under the same conditions. Various chemicals containing 0.01% of a spreading agent (myrinose) were spray-treated at 3 to 5 ml per pot on the Arabidopsis cultivated as described above.

After the treatment, the plate was left to stand at 22° C. under a cycle condition of a light time of 12 hours and a dark time of 12 hours, and sampled after 2 days.

The sampled plant was frozen in liquid nitrogen, and total RNA was extracted using Maxwell ^™ RSC simplyRNA Tissue Kit (Promega). Subsequently, single-stranded cDNA was synthesized from total RNA using PrimeScript RT reagent Kit (Takara). Furthermore, quantitative RT-PCR was performed using SsoAdvanced ^™ Universal SYBR ^™ Green Supermix (manufactured by Bio-Rad). Quantitative RT-PCR was performed using 1×SsoAdvanced ^™ Universal SYBR ^™ Green Supermix, 200 nM reaction mixture of forward primer and reverse primer at 95°C for 10 seconds, followed by denaturation step at 95°C for 5 seconds and 65°C. The cycle of 20 seconds (20 seconds at 60° C. for CBP20) was set as one cycle, and 40 cycles were performed.

The standardization between samples was performed by using the CBP20 gene constitutively expressed in Arabidopsis thaliana and dividing the expression level of the target gene in each sample by the expression level of the CBP20 gene.

The sequences of the primers used for quantitative RT-PCR of each gene in Arabidopsis are as follows.

CBP20 (At5g44200; forward primer 5'-TGTTTCGTCCTGTTCTACTC-3'/SEQ ID NO: 1, reverse primer 5'-CCCATTGTCTTCCTTCTTG-3'/SEQ ID NO: 2).

PR1 (At2g14610; forward primer 5'-CCCACAAGATTATCTAAGGGTTCAC-3'/SEQ ID NO: 3, reverse primer 5'-CCCTCTCGTCCCACTGCAT-3'/SEQ ID NO: 4) (Jirage et al. (2001) Plant J 26: 395-407) ..

PDF1.2 (At5g44420; forward primer 5'-TTGCTGCTTTCGACGCA-3'/SEQ ID NO:5, reverse primer 5'-TGTCCCACTTGGCTTCTCG-3'/SEQ ID NO:6).

The samples were monitored for expression of PR1 gene and PDF1.2 gene by quantitative RT-PCR method. The results are shown in Figure 1.

[Example 2] Inhibitory effect of phytopathogenic fungus on Pak choi that was treated with various metal chelates (1) Inhibition of infection of Brassicaceae plant black spot bacterial disease on Pak choi The infection inhibitory effect against black spot bacterial disease was evaluated by the following method.

As a plant material, a Brassicaceae plant, Pakchoi (variety: Qing, Xiao Pao), was sown under the same conditions as in Example 1, and a 24-hour light-dark cycle (light time 16 hours and dark time 8 hours was performed at 24°C. ). The agent to which 0.1% of the spreading agent (Approach BI) was added was applied to foliage plant which was cultivated for 10 days after sowing. Two days after the drug was sprayed, Pak choi was spray-inoculated with black spot bacterial pathogen (1×10 ⁸ cfu/ml). The inoculated plant was allowed to stand in a moist chamber. Three days after the inoculation, the inoculated leaves were sampled, and the qRT-PCR method was performed in the same manner as in Example 1 to measure the expression level of the rpoD gene (Psm-rpoD) of the black spot bacterial pathogen, and thus the infectious dose to Qing bean plant. Was quantified. However, the RT-PCR reaction consisted of a denaturation step at 95°C for 10 seconds, followed by 5 seconds at 95°C and 20 seconds at 60°C (20 seconds at 65°C for BrACT2) as one cycle, and 40 Went through a cycle. As a control, only the spreading agent containing no metal (0.1% approach BI) was sprayed, and the same quantitative determination was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

The standardization between samples was performed by using the BrACT2 gene constitutively expressed in Pak choi and dividing the expression level of the target gene in each sample by the expression level of BrACT2 gene.

The sequences of the primers used for the quantitative RT-PCR of each gene in Pakchoi are as follows.

Psm-rpoD (forward primer 5'-CCGAGATCAAGGACATCAAC-3'/SEQ ID NO:7, reverse primer 5'-GAGATCACCAGACGCAAGTT-3'/SEQ ID NO:8)
BrACT2 (forward primer 5'-GCCGAGGCTGATGACATT-3'/SEQ ID NO: 9, reverse primer 5'-CATGATGTCTTGGCCTACCA-3'/SEQ ID NO: 10).

As a result, compared with the control, the infection suppressing effect was significantly observed in the metal solution (Fig. 2). No phytotoxicity was observed in the treatment with zinc gluconate (ZnGluc).
(2) Suppression of infection of Brassicaceae plant black spot bacterial disease in Pakchoi (Pakchoi) The effect of inhibiting infection with black spot bacterial disease was evaluated by the following method.

As a plant material, a Brassicaceae bok choy (Pharmaceae) is sown under the same conditions as in Example 1 and cultivated at 24° C. in a 24-hour light-dark cycle (light time 16 hours and dark time 8 hours). did. To the bok choy cultivated for 10 days after sowing, various agents to which 0.01% of a spreading agent (mylino) was added were sprayed on foliage. Two days after the drug was sprayed, Pak choi was spray-inoculated with black spot bacterial pathogen (1×10 ⁸ cfu/ml). The inoculated plant was allowed to stand in a moist chamber. Three days after the inoculation, the inoculated leaves were sampled, and the expression level of the rpoD gene (Psm-rpoD) of the black spot bacterial pathogen was measured by the qRT-PCR method in the same manner as in Example 2(1) to obtain the Pak-choi. The amount of infectious disease was quantified.

As a control, only a spreading agent containing no metal (0.01% mylino added) was sprayed, and the same quantitative determination was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

The standardization between samples was performed by using the BrACT2 gene constitutively expressed in Pak choi and dividing the expression level of the target gene in each sample by the expression level of BrACT2 gene.

As a result, in comparison with the control, the effect of suppressing infection was significantly observed in the metal solution (Fig. 3).

Although chemical damage such as spots was observed only with the chemical treatment in the treatment groups of the copper sulfate (CuSO ₄ ) solution and the zinc sulfate (ZnSO ₄ ) solution, copper gluconate (CuGluc) and zinc gluconate (metal chelate) No phytotoxicity was observed in the treated area of ZnGluc).

(3) Suppression of Infection of Brassicaceae Vegetable Anthracnose in Pakchoi The infection control effect on Brassicaceae vegetable Anthracnose was evaluated by the following method.

As a plant material, Brassicaceae bok choy (cultivar: Xiao Pao) was sown under the same conditions as in Example 1, and cultivated at 24° C. in a 24-hour light-dark cycle (light time 16 hours and dark time 8 hours). did. To the bok choy cultivated for 10 days after sowing, various agents containing 0.01% of a spreading agent (mylino) were sprayed on the leaves. Two days after the spraying of the drug, bok choy was spray-inoculated with anthracnose anthracnose fungus (5×10 ⁵ spores/ml). The inoculated plant was allowed to stand in a moist chamber. Five days after the inoculation, the inoculated leaves were sampled and the expression level of the actin gene (Ch-ACT) in the cruciferous anthracnose of Brassicaceae was measured by the qRT-PCR method in the same manner as in Example 2(1). The amount of infection was determined. However, the RT-PCR reaction was carried out for 40 cycles, with a denaturation step at 95°C for 10 seconds, a cycle of 95°C for 5 seconds and 65°C for 20 seconds as one cycle. As a control, only the spreading agent containing no metal (0.01% mylino added) was sprayed, and the same quantification was performed. The relative infectious pathogen amount when the infectious dose of the control was 100 was shown.

The standardization between samples was performed by using the BrACT2 gene constitutively expressed in Pak choi and dividing the expression level of the target gene in each sample by the expression level of BrACT2 gene.

The sequences of the primers used for the quantitative RT-PCR of each gene in Pakchoi are as follows.

Ch-ACT (forward primer 5'-CTCGTTATCGACAATGGTTC-3'/SEQ ID NO: 11, reverse primer 5'-GAGTCCTTCTGGCCCATAC-3'/SEQ ID NO: 12).

As a result, in comparison with the control, the metal chelate solution showed a significant infection suppressing effect (Fig. 4).

Although chemical damage such as spots was observed only with the chemical treatment in the treatment groups of the copper sulfate (CuSO ₄ ) solution and the zinc sulfate (ZnSO ₄ ) solution, copper gluconate (CuGluc) and zinc gluconate (metal chelate) No phytotoxicity was observed in the treated area of ZnGluc).

[Example 3] Inhibitory effect of plant pathogens on Arabidopsis treated with various metal chelates (1) Inhibition of infection of cruciferous vegetables Anthracnose on Arabidopsis Arabidopsis thaliana (ecotype: Colombia) was used as a plant material in the same manner as in Example 1. It was cultivated under the conditions. One week after sowing, the seedlings were replanted so that there were 5 individuals per pot, and the plants were further cultivated for 3 weeks under the same conditions. Various chemicals containing 0.01% of spreading agent (myrinose) were sprayed on the leaves at 3 to 5 ml per pot. Two days after the drug application, Arabidopsis thaliana was spray-inoculated with anthracnose anthracnose (5×10 ⁵ spores/ml). The inoculated plant was allowed to stand in a moist chamber. Five days after the inoculation, the inoculated leaves are sampled, and the expression level of the actin gene (Ch-ACT) in the cruciferous anthracnose of Brassicaceae is measured by the qRT-PCR method in the same manner as in Examples 1 and 2(3). The amount of infection to Arabidopsis was quantified by. As a control, only the spreading agent containing no metal (0.01% mylino) was sprayed, and the same quantitative determination was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

The standardization between samples was performed by using the CBP20 gene constitutively expressed in Arabidopsis thaliana and dividing the expression level of the target gene in each sample by the expression level of the CBP20 gene.

As a result, compared with the control, the infection suppressing effect was significantly observed in the metal solution (Fig. 5).

In the copper sulfate (CuSO ₄ ) solution treatment group and the zinc sulfate (ZnSO ₄ ) solution treatment group, phytotoxicity such as spots was observed only by the chemical treatment, but metal chelates such as copper gluconate (CuGluc) and gluconate No phytotoxicity was observed with zinc (ZnGluc) or iron gluconate (FeGluc).

(2) Inhibition of infection with cruciferous black spot bacterial pathogen in Arabidopsis thaliana Arabidopsis thaliana (ecotype: Colombia) cultivated in the same manner as in Example 1 was supplemented with various agents containing 0.01% of a spreading agent (myrinose). The foliage was sprayed at 3 to 5 ml per pot. Two days after spraying the drug, Arabidopsis thaliana was spray-inoculated with black spot bacterial pathogen (1×10 ⁸ cfu/ml). The inoculated plant was allowed to stand in a moist chamber. Three days after the inoculation, the inoculated leaves were sampled, and the expression level of the rpoD gene (Psm-rpoD) of the black spot bacterial pathogen was measured by the qRT-PCR method in the same manner as in Examples 1 and 2(1). The amount of infection to Arabidopsis was quantified. As a control, only the spreading agent containing no metal (0.01% mylino added) was sprayed, and the same quantitative determination was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

The standardization between samples was performed by using the CBP20 gene constitutively expressed in Arabidopsis thaliana and dividing the expression level of the target gene in each sample by the expression level of the CBP20 gene.

As a result, compared with the control, the infection suppressing effect was significantly observed in the metal solution (Fig. 6).

In the zinc sulfate (ZnSO ₄ ) solution-treated group, phytotoxicity such as spots was observed only with the chemical treatment, but no phytotoxicity was observed with the metal chelate zinc gluconate (ZnGluc).

[Example 4] Inhibition effect of phytopathogenic fungi on tomatoes treated with various metal chelates (1) Inhibition of infection of chlorophyll bacterial pathogens on tomatoes Tomatoes of the Solanaceae plants (variety: Regina) were used as Example 1 The seeds were sown under the same conditions and cultivated at 24° C. in a 24-hour light-dark cycle (light time 16 hours and dark time 8 hours). Each compound (0.1% approach BI added) was sprayed on the tomatoes cultivated for 3 weeks after sowing. Three days after spraying each compound, Pseudomonas syringae (1×10 ⁸ cfu/ml) was spray-inoculated. Three days after the inoculation, the inoculated leaves were sampled, and the expression level of the rpoD gene (Ps-rpoD) of Pseudomonas syringae (Ps-rpoD) was measured by the qRT-PCR method in the same manner as in Examples 1 and 2(1) to give tomatoes. The amount of infectious disease was quantified. However, the RT-PCR reaction was carried out for 40 cycles with a denaturation step of 95° C. for 10 seconds, followed by a step of 95° C. for 5 seconds and 60° C. for 20 seconds as one cycle. As a control, only the spreading agent containing no compound (0.1% approach BI added) was sprayed and the same quantification was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

Standardization between samples was performed by using the SLTIP41 gene that is constantly expressed in tomato and dividing the expression level of the target gene in each sample by the expression level of the SLTIP41 gene.

The sequences of the primers used for the quantitative RT-PCR of each gene in tomato are as follows.

SLTIP41 (forward primer 5'-ATGGAGTTTTTGAGTCTTCTGC-3'/SEQ ID NO: 13, reverse primer 5'-GCTGCGTTTCTGGCTTAGG-3'/SEQ ID NO: 14).

As a result, in comparison with the control, the metal chelate solution showed a significant infection suppressing effect (Fig. 7).
(2) Suppression of infection by Botrytis cinerea in tomato The effect of inhibiting infection by Botrytis cinerea on tomato was evaluated by the following method.

Each compound (0.1% approach BI added) was sprayed on tomatoes by foliage. Three days after spraying each compound, spores of Tomato Botrytis cinerea were suspended in 1/2 potato decoction medium (2×10 ⁵ cells/mL), and the tomato strain was spray-inoculated. Six days after the inoculation, the infectious dose of gray mold of tomato was evaluated by investigating the symptom. As a control, only the spreader containing no compound (0.1% approach BI added) was sprayed and the same evaluation was performed.

The degree of disease is represented by the following formula.
Disease incidence = {(1n ₁ +2n ₂ +3n ₃ +4n ₄ +5n ₅ )/(5 x number of surveys)} x 100
In the disease onset survey, the degree of onset was divided into the following five categories.
0: No symptom, 1: Spots are found in a small part of the surveyed strains, 2: Spots are found in less than 1/3 of the surveyed strains, 3: 1/3 or more and less than 2/3 of the surveyed strains Lesions are observed in 4: 4, lesions are observed in 2/3 or more of the investigated strains, 5: death
n ₁ to n ₅ are the number of individuals and the control value is expressed by the following formula.
Control value={1-the degree of disease in the treated area/the degree of disease in the untreated area)}×100
As a result, in comparison with the control, the effect of suppressing infection was significantly observed in the metal solution (Table 1).

Example 5 Inhibitory Effect of Plant Pathogens on Cucumbers Treated with Various Metal Chelates Each compound (0.1% Approach BI added) was sprayed onto cucumber (variety: New Hokusei). Three days after the application of each compound, spray-inoculation was performed with an anthracnose fungus (5x10 ⁵ cells/mL). Seven days after the inoculation, the inoculated leaves were sampled, and the qRT-PCR method was used in the same manner as in Example 1 to measure the expression level of the actin gene (Co-ACT) of the Cucurbital anthracnose bacterium, thereby infecting the cucumber. Was quantified. However, the RT-PCR reaction consisted of a denaturation step at 95°C for 10 seconds, followed by 5 seconds at 95°C and 20 seconds at 65°C (20 seconds at 60°C for CsEF1α) as one cycle. Went through a cycle. As a control, only the spreading agent containing no compound (0.1% approach BI) was sprayed and the same quantification was performed. The relative infectious pathogen amount when the control infectious amount was 100 is shown.

Standardization between samples was performed by using the CsEF1α gene that is constitutively expressed in cucumber and dividing the expression level of the target gene in each sample by the expression level of the CsEF1α gene.

The sequences of the primers used for the quantitative RT-PCR of each gene in cucumber are as follows.

Co-ACT (forward primer 5'-CTCGTTATCGACAATGGTTC-3'/SEQ ID NO:15, reverse primer 5'-GAGTCCTTCTGGCCCATAC-3'/SEQ ID NO:16).

CsEF1α (forward primer 5′-ACTGTGCTGTCCTCATTATTG-3′/SEQ ID NO: 17, reverse primer 5′-AGGGTGAAAGCAAGAAGAGC-3′/SEQ ID NO: 18).

As a result, in comparison with the control, the metal chelate solution showed a significant infection suppressing effect (Fig. 8).

[Example 6] Inhibitory effect of phytopathogenic fungi on strawberries treated with various metal chelates (1) Inhibitory effect of anthracnose fungus infection on strawberries The inhibitory effect on strawberry anthracnose fungus (Colletotrichum fructicola) was evaluated by the following method.

Strawberries (variety: Onamine) were sprayed with each compound (0.1% Approach BI added). Three days after spraying each compound, strawberry anthracnose (5×10 ⁵ cells/mL) was spray-inoculated. Six days after the inoculation, the amount of strawberry B. anthracis infectious to the strawberry was evaluated by investigating the symptoms. As a control, only the spreader containing no compound (0.1% approach BI) was sprayed and the same evaluation was performed.

The degree of disease is represented by the following formula.
Disease incidence = {(1n ₁ +2n ₂ +3n ₃ +4n ₄ +5n ₅ )/(5 x number of surveys)} x 100
In the disease onset survey, the degree of onset was divided into the following five categories.
0: No symptom, 1: Small spots, 2: Lesion is observed on less than 25% of the leaf area, 3: Lesion is found on more than 25% and less than 50% of leaf area, 4: Leaf Lesions are observed in 50% or more of the area or petiole is broken, 5: Death
n ₁ to n ₅ are the number of individuals and the control value is expressed by the following formula.
Control value={1-the degree of disease in the treated area/the degree of disease in the untreated area)}×100
As a result, in comparison with the control, the effect of suppressing infection was significantly observed in the metal solution (Fig. 9).

In addition, the chemical treatments of copper sulfate (CuSO ₄ ), copper acetate (CuOAc), and zinc sulfate (ZnSO ₄ ) showed chemical damages such as spots, but the metal chelate copper gluconate (CuGluc ₄ ) was observed. ), zinc gluconate (ZnGluc), and zinc glycine did not show any phytotoxicity.

(2) Inhibition of powdery mildew infection in strawberry The following method was used to evaluate the effect of suppressing infection of strawberry powdery mildew.

Strawberry seedlings (cultivar: Onamine) (about 1 colony per compound leaf) in which slight strawberry powdery mildew had begun were sprayed with each compound (0.1% approach was added). Seven days after the spraying, the infection amount of strawberry powdery mildew fungus on strawberry was evaluated by investigating the symptom. As a control, only a spreading agent containing no compound (0.1% approach added) was sprayed and the same evaluation was performed. As a result, in comparison with the control, the effect of suppressing infection was significantly observed in the metal solution (Table 2).

[Example 7] Inhibitory effect of plant pathogens on Chinese cabbage treated with various metal chelates After planting a test plant (Chinese cabbage variety: Musou), it was cultivated for 14 days. 4 mM copper gluconate and 4 mM zinc gluconate (with 0.1% approach BI added) were applied to the foliage, and 3 days after the application, the seedlings were inoculated with 1×10 ⁵ cells/ml of the Chinese cabbage soft rot fungus (Erwinia carotovora subsp. carotovora). .. Six days after the inoculation, the degree of illness was investigated. The control value was calculated from the degree of disease onset.
The degree of disease is represented by the following formula.
Disease incidence = {(1n ₁ +2n ₂ +3n ₃ +4n ₄ )/(4 x number of surveys)} x 100
In the disease onset survey, the degree of onset was divided into the following four categories.
0: No symptom, 1: Lesion is observed on less than 5% of the leaf area, 2: Lesion is found on 5% or more and less than 25% of leaf area, 3: 25% or more and 50% of leaf Lesions are observed in areas of less than 4, 4: lesions are observed in more than 50% of the leaves
n ₁ to n ₄ are the number of individuals and the control value is expressed by the following formula.
Control value={1-the degree of disease in the treated area/the degree of disease in the untreated area)}×100
As a result, each of the above compounds showed a control value of 30% against soft rot (Table 3).

[Example 8] Inhibitory effect of phytopathogenic bacteria on Bensamiana tobacco treated with various metal chelates A solution containing copper gluconate, zinc gluconate, and iron gluconate in Bensamyana tobacco (Solanaceae plant) 4 weeks after sowing Was sprayed on the leaves. Three days after the treatment, a droplet (10-20 μL) of a zoospore suspension containing 1000 zoospores of Phytophthora infestans was instilled. Symptoms were examined 5 days after the inoculation. As a result, compared with the control group, the chelate solution treatment group significantly suppressed the disease onset.

[Reference Example 1] Relationship between control effect against plant viral disease and control effect against phytopathogenic fungi From a phytopathological standpoint, viruses and pathogenic fungi (filamentous fungi, bacteria) have different infection behaviors against plants. Even if there is a controlling effect against plant viral diseases, it is considered unpredictable to a person skilled in the art whether or not it has a controlling effect against phytopathogenic fungi.

In order to verify this, the present inventor verified the control effect of lentemin, which is the only pesticide for controlling plant viral diseases, against phytopathogenic fungi.

Using a 10-fold diluted lentemin solution (0.1% approach BI added), the same evaluation as in Example 6(1) was performed, and lentemin did not suppress the infection with B. anthracis, and rather than the control. It became severely infected (Table 4).

In addition, when Z bordeaux (containing copper) or Geozette (containing zinc), which are commercially available as fungicides, were applied to Bensamiana tobacco, these agents did not suppress the infection of tomato mosaic virus (ToMV) ( Data not shown).

These results support that there is no direct correlation between the control effect of a drug on plant viral diseases and the control effect on plant pathogens.

In this sense, it is surprising and surprising that the metal chelate (Patent Document 1), which is known to have a controlling effect against plant viral diseases, also showed a controlling effect against plant pathogenic fungi in this example. there were.

As described above, the control agent of the present invention can exhibit an excellent control effect against plant pathogens. By using a metal chelate as an active ingredient, it is possible to exert a controlling effect against phytopathogenic fungi without causing phytotoxicity such as leaf atrophy and spotting, and thus as an agricultural chemical having both safety and effect, It can greatly contribute to the agricultural field.

Claims (4)

A control agent for phytopathogenic fungi, which contains at least one of zinc gluconate, zinc glycine, and iron gluconate as an active ingredient. The control agent according to claim 1, wherein the phytopathogen is a phytopathogenic bacterium or a phytopathogenic fungus. A method for controlling phytopathogenic fungi, which comprises applying the control agent according to claim 1 to a plant. The control method according to claim 3 , wherein the phytopathogenic fungus is a phytopathogenic bacterium or a phytopathogenic fungus.