WO2014136967A1 - 育苗期病害耐病性植物種子の製造方法及び育苗期病害の発病予防及び防除方法 - Google Patents
育苗期病害耐病性植物種子の製造方法及び育苗期病害の発病予防及び防除方法 Download PDFInfo
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- WO2014136967A1 WO2014136967A1 PCT/JP2014/056059 JP2014056059W WO2014136967A1 WO 2014136967 A1 WO2014136967 A1 WO 2014136967A1 JP 2014056059 W JP2014056059 W JP 2014056059W WO 2014136967 A1 WO2014136967 A1 WO 2014136967A1
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- seedling
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
- A01H5/10—Seeds
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
Definitions
- the present invention relates to a method for producing seedling disease-resistant plant seeds using non-pathogenic bacteria corresponding to seed infectious pathogens, and to prevent the occurrence of seedling disease in the next generation using seedling disease-resistant plant seeds. , Relating to control technology.
- Plant seed infectious diseases are positioned as seedling diseases as well as soil infectious diseases.
- the seed infectious pathogen causing a seed infectious disease is often transmitted to the next generation by the carrier-bearing seed and spreads by infecting the seedling plant of the next generation.
- a rice seedling disease is an agriculturally important seed-borne disease of rice caused by a rice seedling fungus belonging to Fusarium fujikuroi (full generation name Gibberella fujikuroi).
- Fusarium fujikuroi full generation name Gibberella fujikuroi
- the pathogenic bacteria are not only transmitted to surrounding healthy rice seedlings, but the infected plants show abnormal length and yellowing, and then die. It reaches.
- many conidia of this fungus are formed on the lower surface of the leaf sheath, and these conidia are scattered and adhere to the flower parts, pistils, cocoons, and carcasses of surrounding healthy strains, and the cocoon is contaminated.
- the cocoon becomes a transmission source for the following year as a germ-bearing seed (Non-patent Document 1).
- This disease can be effectively controlled by seed disinfection with chemical pesticides such as benomyl and pefrazoate, and has been calmed down for a long time in the field.
- chemical pesticides such as benomyl and pefrazoate
- excessive reliance on chemical pesticides hastened the emergence of resistant bacteria, raising concerns about the invalidation of these drugs.
- the disease was calmed down by using bactericides such as ipconazole with different mechanisms of action.
- the use of chemical pesticides always presents a risk of the appearance of resistant bacteria, and problems such as environmental pollution and residue on rice remain.
- the conventional physical control method and biological control method are unstable compared to chemical pesticides and cannot sufficiently suppress the occurrence of seed infectious diseases such as rice seedling disease.
- the labor force for processing increased.
- problems in terms of cost there were problems in the case of microbial pesticides, there were problems in terms of cost, and in the case of physical control methods, there were problems such as complexity of treatment and a decrease in seed germination rate.
- An object of the present invention is to provide a safe and inexpensive organism that has a high control effect on seedling diseases including seed infectious diseases and soil infectious diseases for stable food production and supply.
- seedling diseases including seed infectious diseases and soil infectious diseases for stable food production and supply.
- microbial pesticides By developing microorganisms and providing microbial pesticides using them as active ingredients, establishing effective treatment methods for the microbial pesticides, and effectively controlling seedling diseases It is to provide technology that can reduce the labor and cost and stably grow safe agricultural products.
- the biological control method using a microbial pesticide has a big problem that its effect is not stable.
- One of the causes is difficulty in establishing on the plant body, in the tissue, in the rhizosphere, etc., until the microorganisms which are active ingredients exhibit the control effect.
- the present inventors treated a non-pathogenic bacterium corresponding to a seed infectious pathogen and having a plant-fixing property on a flower part of a host plant at the flowering stage by a method such as spraying in advance.
- a method such as spraying in advance.
- a step of bringing a non-pathogenic bacterium corresponding to a seed infectious pathogen into contact with a flower part of a host plant before and after the flowering period, and a step of recovering a seed of the host plant to which the non-pathogenic bacterium obtained after the step is established A method for producing seedling disease-resistant plant seeds, comprising:
- the non-pathogenic bacteria are Fusarium, Nectria, Gibberella, Calonectria, Hypomyces, Trichoderma, Penicillium, Talaromyces, Acremonium, Alternaria, Verticillium
- the non-pathogenic bacterium is a Fusarium genus selected from the group consisting of Fusarium oxysporum, Fusarium moniliforme, Fusarium fujikuroi, Fusarium proliferatum, and Fusarium sacchari, according to any one of (1) to (3) Production method.
- Seeds of disease-resistant plants at the seedling stage obtained by contacting non-pathogenic bacteria corresponding to seed-borne pathogens with the flower parts of the host plant before and after the flowering period.
- a microbial pesticide for controlling diseases during the seedling stage comprising a non-pathogenic bacterium corresponding to a seed infectious bacterium as an active ingredient.
- seedling disease-resistant plant seeds of the present invention it is possible to produce and provide seedlings (seedlings) resistant to seedling disease.
- plant seeds resistant to seedling disease can be easily and efficiently produced.
- the method for preventing and controlling the seedling disease of the present invention it is possible to prevent and control the disease of the seedling disease.
- an inexpensive and highly efficient microbial pesticide for efficiently preventing and controlling the seedling disease can be provided.
- the first aspect of the present invention relates to a method for producing seedling disease-resistant plant seeds (often referred to herein as “manufacturing method”). According to the production method of the present invention, it is possible to produce and provide a plant seed that has acquired disease resistance as if it were a resistant variety against a seedling disease by utilizing a biological control material microorganism.
- the seedling disease refers to a plant disease that is infected or develops during the seedling season, and includes seed infectious diseases and soil infectious diseases.
- “Seed-borne disease” is a plant disease caused by infection with a seed-borne pathogen, and is transmitted to not only the plant strain but also surrounding strains through seeds that have established the seed-borne pathogen during soaking or germination. It is.
- a “soil-borne disease” is a plant disease that is originally caused by infection with pathogenic bacteria that survive in soil.
- “Seed-infectious pathogen” refers to a microorganism that infects a flower part formed in a host plant, then settles on a seed and causes a disease symptom to a seedling individual.
- the “seedling individual” refers to a seedling germinated from a seed and a plant individual from which the seedling has grown.
- the “microorganism” usually refers to a micro organism that is difficult to recognize with the naked eye, such as a bacterium or a bacterium.
- a fungus is a group of eukaryotes belonging to the kingdom of kingdom (Kingdom of fungi) in terms of biological classification, and is sometimes called a fungus or a filamentous fungus.
- the fungus includes a unicellular eukaryotic microorganism such as yeast, or a filamentous fungus (including fungi) or a multicellular eukaryotic microorganism such as mushroom that is relatively difficult to recognize with the naked eye.
- the seed infectious pathogen is a group having pathogenicity included in a specific microbial species as described above.
- microorganisms including seed infectious pathogens include bacteria of the genus Bacillus, bacteria of the genus Pseudomonas (including bacteria of the genus Burkholderia and Acidovorax), bacteria of the genus Xanthomonas, bacteria of the genus Streptomyces, etc. Examples include Fusarium, Nectria, Gibberella, Calonectria, Hypomyces, Trichoderma, Penicillium, Talaromyces, Acremonium, Alternaria, Verticillium, and the like. Examples of the genus Fusarium include F. oxysporum, F.
- the genus Fusarium is a major incomplete generation name of the genus Gibberella, and the genus Gibberella is a synonym of the genus Fusarium in this specification.
- the flower part is infected at the flowering stage of the host plant, and then the seed and / or ovary (Poaceae) In the case of fruit, it will settle. Seed-borne pathogens germinate in the seeds that carry them, and settle in plant tissues and rhizosphere along with the growth of seedlings. Causes an infectious disease.
- seed infectious diseases include rice sapling seedling disease (by Gibberella fujikuroi), rice blast disease (by Magnaporthe oryzae), seedling blight (by Fusarium spp., Pythium spp., Rhizoctonia spp., Trichoderma spp.) , Sesame leaf blight (by Cochliobolus miyabeanus), seedling blight (Pseudomonas plantarii (now Burkholderia plantarii)), rice blast blight (Pseudomonas glumae (current Burkholderia glumae)), brown stripe (Pseudomonas avenae (current) Acidovorax avenae)).
- rice sapling seedling disease by Gibberella fujikuroi
- rice blast disease by Magnaporthe oryzae
- seedling blight by Fusarium spp., Pythium spp., Rh
- the seed plants for infectious pathogens include angiosperms and gymnosperms.
- Angiosperms may be either dicotyledonous plants or monocotyledonous plants.
- Monocotyledonous plants include, for example, Poaceae plants.
- Examples of dicotyledonous plants include Rosaceae plants, Solanaceae plants, Fabaceae plants, Cucurbitaceae plants, Brassicaceae plants, and the like.
- it is a gramineous plant.
- the grasses in this specification include, for example, rice (Oryza sativa and O. glaberrima), wheat Triticum aestivum, T. compactum, and T.
- durum barley (Hordeum vulgare), rye (Secale cereale), millet ( Agriculturally important species such as Panicum miliaceum, millet (Setaria italica), millet (Echinochloa esculenta), sorghum (Sorghum bicolor), corn (Zea mays), and sugarcane (Saccharum officinarum) are included.
- Preferred are rice, wheat, barley, corn and sugar cane, and more preferred is rice.
- Bio control material microorganism is synonymous with “non-pathogenic fungus corresponding to seed infectious pathogen” in the present specification.
- the term “corresponding” as used herein means that they are mainly of the same kind, the same genus or closely related to each other in taxonomy, or have a biological control effect. Therefore, as described above, “non-pathogenic fungi corresponding to seed infectious pathogens” refers to a group that is taxonomically the same species as a seed infectious pathogen but has no pathogenicity. Say. For example, in the case of a non-pathogenic bacterium corresponding to a rice sapling seedling fungus, a non-pathogenic bacterium of the genus Fusarium, for example, F.
- the biological control material microorganism has a characteristic of not showing disease symptoms to the host plant while maintaining high infectivity and colonization of the host plant.
- a host plant in which a biological control material microorganism is established becomes resistant to not only seed infectious diseases but also all seedling diseases including soil infectious diseases.
- Specific examples of the biological control material microorganism include Fusarium oxysporum non-pathogenic strain W3 (accession number: NITE BP-01538) or Fusarium oxysporum non-pathogenic strain W5 (accession number: NITE BP-01539).
- Plant disease-resistant plants are plants in which microorganisms for biological control are established, exhibiting disease resistance against all seedling diseases, including seed-borne diseases and soil-borne diseases, and those diseases A plant that does not show clear symptoms.
- the source of the biological control material microorganisms is the plant tissue (leaf, hypocotyl, leaf sheath) of the same homogenous strain that grows around the plant strain affected by the disease in the field where the seed infectious disease occurs. , Petiole, stem, flower vase, fruit, root, etc.) and its rhizosphere soil.
- the plant tissue is used as a separation source, it is preferable to disinfect the tissue surface.
- about 1 cm 2 of the collected plant tissue piece may be immersed in 70% ethanol for 30 seconds, then in 1% hypochlorous acid for 3 minutes to sterilize the tissue surface, and then washed with sterile water.
- concentrations of ethanol and hypochlorous acid and the immersion time are not particularly limited, and may be appropriately adjusted based on a technique known in the art depending on the site of the plant tissue.
- the biological control material microorganism is a fungus
- the plant tissue after surface sterilization is cut into about 1 cm 2 tissue pieces using sterilized tweezers, scalpels, etc. and placed on the medium.
- Medium is PSA (potato decoction agar: 200 g / L potato decoction, 0.5% [w / v] sucrose, 1.5% [w / v] agar) or WA (elemental agar: 1% agar) Use it.
- a selective medium suitable for the genus can be used. In order to prevent bacterial contamination, it is desirable to apply 20% lactic acid in advance on the medium.
- the medium on which the plant tissue is placed is cultured at 25-30 ° C. for 2-5 days.
- Establish a single colony strain by re-implanting a single colony (in the case of single-cell eukaryotic microorganisms), mycelial tips (in the case of filamentous fungi or basidiomycetes), spores or conidia grown on a medium plate into a new medium plate .
- the biological control material microorganism is a bacterium
- the plant tissue after surface sterilization is cut into about 1 cm 2 tissue pieces using sterilized tweezers, a scalpel, etc. and placed on the medium.
- the medium is PSA or King B medium (2% [w / v] peptone, 1% [w / v] glycerin, 0.15% [w / v] dipotassium hydrogen phosphate, 0.15% [w / v] magnesium sulfate.
- a selective medium suitable for the genus can be used.
- the medium on which the plant tissue is placed is cultured at 25-30 ° C. for 1-3 days.
- the generated single colony is scraped off and transplanted to a new medium plate, and the regrown colony is established as a single strain.
- the seed production method for a disease-resistant plant of the seedling stage of the present invention includes a contact step and a recovery step. Hereinafter, each step will be specifically described.
- the “contacting step” is a step of bringing a biological control material microorganism into contact with the flower part of the host plant before and after the flowering period.
- any biological control material microorganism is settled on the host plant.
- the “flowering period” refers to the period during which the host plant is in bloom.
- “Before and after the flowering period” refers to a period including before the start of flowering of the host plant and after the end of flowering. This period is 2 weeks before and after the flowering period, preferably 10 days before and after, more preferably 1 week before and after or 5 days before and after.
- the host plant is rice and it is two weeks before and after the flowering period, it corresponds to the period from the ear development stage where the young ears grow rapidly in rice to the time the seedling matures after the flowering period in the strain is completed To do.
- the time when the biological control material microorganisms are brought into contact with the flower part of the host plant is before the seed infectious pathogens occurring or possibly occurring in the predetermined area are infected with the host plant.
- the biological control material microorganism infects the host plant due to an action (competitive action) that eliminates the infection of other microorganisms by the seed-borne pathogen. This is because the effect of the present invention cannot be obtained.
- the type of biological control material microorganisms to be contacted is not particularly limited. In general, if any biological control material microorganism infects an uninfected host plant, it will be occupied in the host plant by occupying and competing with the infected biological control material microorganism and / or due to the infection of the biological control material microorganism. Induced resistance makes it difficult to infect other seed-borne pathogens.
- the present invention utilizes this principle, and by bringing a biological control material microorganism into contact with a host plant, regardless of the microbial species, a pathogenic bacterium of a seedling stage disease including a seed infectious disease is subsequently infected. Can be controlled.
- the seeds of the host plant obtained by this production method can be resistant to seedling diseases.
- a non-pathogenic bacterium corresponding to a seed infectious pathogen that has occurred or is likely to occur in an area where seeds of a disease-resistant disease-resistant plant should be sown may be used.
- a non-pathogenic fungus of the genus Fusarium corresponding to the rice sapling seedling fungus may be used as a biological control material microorganism.
- Fusariumiumoxysporum W3 strain (accession number: NITE BP-01538) or Fusarium oxysporum W5 strain (accession number: NITE BP-01539) which is a non-pathogenic bacterium derived from the genus Fusarium can be used.
- NITE BP-01538 or Fusarium oxysporum W5 strain which is a non-pathogenic bacterium derived from the genus Fusarium
- NITE BP-01539 which is a non-pathogenic bacterium derived from the genus Fusarium
- the biological control material microorganism to be contacted is not a mixture of different plural bacterial species, but a single bacterial species is contacted.
- the number of contact of the biological control material microorganisms is not limited. In a species in which florets contained in spikelets continuously bloom, such as a gramineous plant, this step may be performed several times in order to infect all the flower parts with the biological control material microorganisms. However, it is desirable that the biological control material microorganisms to be contacted each time are the same strain for the reasons described above.
- the method for contacting the biological control material microorganism with the host plant is not particularly limited as long as it can contact the biological control material microorganism. Examples thereof include a method of spraying, spraying, applying, and immersing a culture solution, suspension, powder, etc. of a biological control material microorganism described later.
- the place of contact with the host plant may be either a part or the whole of the host plant, but when contacting with a part of the plant, a site related to the host plant infection route of the biological control material microorganism, That is, it should be noted that the flower part is brought into contact.
- the “recovery step” is a step of recovering the seeds of the host plant to which the biological control material microorganism obtained after the contact step has been established.
- the biological control material microorganism that has come into contact with the flower part of the host plant in the contacting step usually settles in the cocoon, seed coat or ovule tissue.
- the host plant develops seeds with established non-pathogenic mutants after pollination. Therefore, in this step, seeds that are sufficiently matured to have germination ability may be collected by a method known in the art.
- the seeds obtained from the host plant after the contacting step can in principle be regarded as seeds of a disease-resistant plant at the seedling stage.
- seeds may be dried as necessary to improve the storage stability.
- the drying method may be any method as long as it retains the germinating ability of the seed and the moisture in the seed is moderately reduced as long as the established biological control material microorganism does not die.
- a natural drying method that exposes to the outside air a dehumidifying and drying method that puts the product in a sealed container together with a dehumidifying agent, a wind drying method that uses hot air or cold air to dry using a blower or the like, or a combination thereof.
- Subsequent seed storage methods may follow methods well known in the art.
- the seed after the recovery process is a colonized seed with a biological control material microorganism.
- the base sequence unique to the contacted biological control material microorganism gene is designed as a primer, and it is easily confirmed based on the technology by nucleic acid amplification methods such as PCR. do it.
- the biological control material microorganism is Fusarium oxysporum, it can be a unique base sequence that can identify whether or not the ribosomal DNA IGS region is a biological control material microorganism. Therefore, it may be used as a primer set for amplifying the region.
- FIGs11 (5'-GTAAGCCGTCCTTCGCCTCG-3 ': sequence number 1) and FIGS12 (5'-GCAAAATTCAATAGTATGGC-3': sequence number 2) are mentioned, for example.
- NITE BP-01538 the DNA fragment having the base sequence described in SEQ ID NO: 3
- the Fusarium oxysporum W5 strain accesion No: NITE BP- 01539
- a DNA fragment having the base sequence set forth in SEQ ID NO: 4 is amplified. Therefore, it is possible to confirm and identify which biological control material microorganism derived from Fusarium oxysporum is established.
- the seedlings of the seeds of the disease-resistant disease-resistant plants thus obtained are colonized seeds of the biological control material microorganisms, so that the disease control disease microorganisms develop disease symptoms due to the competitive action of the biological control material microorganisms. It can grow without doing.
- the seeds of the seedlings can easily produce plant seeds that are resistant to any seedling disease as if they were seedling-resistant disease varieties. Can be manufactured and provided.
- the seed production method of the seedling disease-resistant plant of the present invention only needs to be performed at least on the plant for seeding, it is possible to limit the application area of the biological control material microorganism to a minimum, Therefore, the environmental impact is small and economical.
- the seed of the seedling disease-resistant plant of the present invention it is not necessary to carry out the control treatment or the disease suppression treatment for the seed infectious disease on the seed before sowing, and the host that does not cause the seedling disease only by sowing as usual Since plants can be cultivated, labor and costs can be reduced. In addition, seed disinfection with chemical pesticides is unnecessary or can be reduced.
- the seed production method of the disease-resistant plant of the seedling stage of the present invention is highly safe because it is a microbial pesticide using non-pathogenic bacteria existing in nature, and has less impact on the environment than chemical pesticides.
- the disease prevention and control method of the seedling stage disease The second aspect of the present invention relates to the disease prevention and control method of the seedling stage disease.
- ADVANTAGE OF THE INVENTION According to this invention, the onset of the seedling stage disease in the seedling seedling seedling seedling seedling seedling seedling seedling seedling and other plant individuals around the seedling seedling can be prevented and controlled.
- the seedling stage disease onset method of the present invention (hereinafter, often referred to as “preventive control method” in the present specification) is the seedling stage disease resistant plant according to the first aspect in which a biological control material microorganism is established. Use seeds.
- the basic process of the preventive and control method of the present invention is only to carry out a normal cultivation method for plant species of seedling disease-resistant plants. However, seed disinfection performed prior to sowing is not necessary.
- seed seeds of seedling disease and disease resistant rice are germinated and seeded in a container (seedling box, etc.) containing culture soil to raise seedlings. After that, it is sufficient to plant the seedlings in the field and cultivate them by the usual method.
- the seed of the seedling disease-resistant plant described in the first aspect is settled with a biological control material microorganism.
- the biological control material microorganism is a non-pathogenic bacterium, it does not cause disease at the seedling stage. Therefore, the seedling can efficiently prevent and control the infection of pathogenic bacteria of seedling diseases including seed infectious diseases and soil infectious diseases.
- the biological control material microorganisms that have settled on seeds of seedling-borne disease-resistant plants are non-pathogenic bacteria but retain their infectivity. When cultivated together with seeds that are not infected with pathogenic fungi, they can be transmitted to surrounding healthy seedlings during the soaking and seedling periods. However, this results in the occupation of the biological control material microorganisms and resistance to pathogens induced by the infected plants themselves, and the subsequent infection of pathogens of seedling diseases including seed-borne diseases and soil-borne diseases is efficient. Can be prevented and controlled.
- seedling diseases including seed infectious diseases and soil infectious diseases can be efficiently prevented and controlled without requiring special treatment.
- seed infectious diseases and soil infectious diseases are observed not only in seedling seedling seedling seedling seedling seedling seedling seedling seedling seedlings as described in the first aspect, but also in healthy seedling individuals cultivated therewith. It is possible to efficiently prevent and control the infection of pathogenic bacteria of the seedling stage disease.
- a microbial pesticide for controlling seedling diseases A third aspect of the present invention relates to a microbial pesticide for controlling diseases in the seedling season.
- the microbial pesticide for seedling stage disease control of the present invention can be applied to a desired plant to impart seedling stage disease resistance to the plant.
- the biological control material microorganism described in the first aspect may be used as the biological control material microorganism.
- Specific examples of biological control material microorganisms include, for example, Fusarium oxysporum W3 strain (accession number: NITE BP-01538) or Fusarium oxysporum W5 strain (Fusarium oxysporum W5 strain) Accession number: NITE BP-01539).
- NITE BP-01538 Fusarium oxysporum W3 strain
- Fusarium oxysporum W5 strain Feusarium oxysporum W5 strain Accession number: NITE BP-01539
- the amount of the biological control material microorganism per predetermined amount of the microbial pesticide for controlling seedling diseases of the present invention depends on various conditions such as the type, the type of plant to be applied, the dosage form, and the application (contact) method. .
- the biological control material microorganism of the active ingredient contains a sufficient amount to contact and settle the application target plant. This amount is determined within the range of common technical knowledge in the field, taking into account each condition so that the biological control material microorganisms contained in the microbial pesticide of the present invention have a desired amount for the target plant after application. What is necessary is just to determine content of material microorganisms.
- the biological control material microorganism when the biological control material microorganism is derived from the genus Fusarium and the microbial pesticide of the present invention is in a liquid state, its conidia is 1.0 ⁇ 10 4 pieces / mL or more, preferably 5.0 ⁇ 10 4 pieces / mL in the solution. It should be contained more than mL.
- the upper limit is not particularly limited, but 1.0 ⁇ 10 9 cells / mL is usually sufficient even for a biological control material microorganism with a low infection rate.
- the microbial pesticide of the present invention can contain a carrier that is acceptable in an agrochemical formulation as long as it does not inhibit or suppress the host plant infection activity of the biological control material microorganism.
- “Agrochemically acceptable carrier” is a substance that facilitates the application of microbial pesticides, maintains the viability and infectivity of the active biological control material microorganism, and / or controls the action rate of microbial pesticides. In addition, it refers to a substance that has no or little harmful effect on the environment such as soil and water quality even when applied to the cultivation of plants including outdoors and indoors, or has no or low harmfulness to animals, particularly humans.
- an excipient can be mentioned. Suitable excipients include ground natural minerals, ground synthetic minerals, emulsifiers, dispersants and surfactants.
- ground natural minerals examples include kaolin, clay, talc and chalk.
- Examples of the pulverized synthetic mineral include highly dispersed silica and silicate.
- Examples of the emulsifier include nonionic emulsifiers and anionic emulsifiers (for example, polyoxyethylene fatty alcohol ethers, alkyl sulfonates, and aryl sulfonates).
- dispersant examples include lignosulfite waste liquor and methylcellulose.
- surfactant examples include lignosulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid alkali metal salt, alkaline earth metal salt and ammonium salt, alkylaryl sulfonate, alkyl sulfate, alkyl sulfonate, fat Alcohol sulfates, fatty acids and sulfated fatty alcohol glycol ethers, sulfonated naphthalene and naphthalene derivatives and formaldehyde condensates, naphthalene or naphthalenesulfonic acid and phenol and formaldehyde condensates, polyoxyethylene octylphenyl ether, ethoxylated isoforms Octylphenol, octylphenol, nonylphenol, alkylphenyl polyglycol ether, tributylphenyl polyglycol
- the microbial pesticide of the present invention can include one or more carriers that are acceptable in the agrochemical formulation.
- other active ingredients having other pharmacological effects that is, herbicides, fungicides, insecticides, fertilizers (for example, urea, ammonium nitrate, superphosphate), as long as they do not affect the infection of microorganisms. Salt).
- the dosage form of the microbial pesticide of the present invention may be in any state as long as it can maintain the host plant colonization of the biological control material microorganism.
- the biological control material microorganism is suspended in an appropriate solution. It can be a turbid liquid state, a solid state (including a powder state), or a combination thereof.
- the biological control material microorganism which is an active ingredient may be suspended in an appropriate solution.
- Appropriate solutions include, for example, water, buffers, and culture media for their biological control material microorganisms.
- the solid state there is no particular limitation as long as the biological control material microorganism as an active ingredient can act on the host plant.
- Examples thereof include a granular state, a powder state, and a semi-solid state such as a gel. Considering that it adheres to the host plant by contact or the like, and grows and acts, it is preferably in the form of powder (particularly powder having adhesiveness) or gel.
- Example 1 Effect of disease control in seedling disease-resistant plants (1) (the purpose) Preparation of a microbial pesticide for controlling seedling diseases of the present invention, preparation of seeds by a method for producing seedling disease-resistant plant seeds using the same, and seedling seedlings of which are seed-resistant to seedling diseases was verified on a pot scale.
- Fusarium oxysporum non-pathogenic fungus W3 strain (Accession No .: NITE BP-01538) and W5 strain (Accession No .: NITE BP-01539) were used.
- Each biological control material microorganism was cultured in PSB (potato decoction) medium (200 g / L potato decoction, 0.5% [w / v] sucrose) with shaking at 28 ° C. for 5 days. Collect conidia in the culture solution by centrifugation (1,500 ⁇ g, 20 minutes), and spore suspension diluted to 1.0 ⁇ 10 5 to 1.0 ⁇ 10 7 cells / mL in sterile water. It was a microbial pesticide for control.
- PSB potential decoction
- Rice (variety: Tan Ginbo) was used as the host plant. After irrigating by putting 80 g of sterilized culture soil as a floor soil in a 200 mL plastic pot, the two rice grains were sown as parent rice, covered with 20 g of sterilized culture soil, and irrigated again. After sowing, the plants were cultivated for 100 days in an artificial weather room set at 26 ° C under natural light conditions.
- the method of bringing the microbial pesticide into contact with the parent rice is as follows.
- the microbial pesticide is flowered using a hand spray in the morning (around 10:00 to 11:00) when the rice blooms. Sprayed directly on the part (contact process).
- the spray amount was about 5 mL per ear.
- the parent rice after the spray treatment was cultivated until the seeds matured in an artificial weather chamber set at 26 ° C under natural light conditions. When the above-ground part of the parent rice turned yellow, threshing was carried out to collect seed meal (recovery process).
- the seed pods collected by this treatment are hereinafter referred to as “W3-treated seed pods” and “W5-treated seed pods” (corresponding to seeds of the disease-resistant plant at the seedling stage of the present invention).
- the control seed potato collected by the same operation except that sterilized water is sprayed instead of the microbial pesticide is hereinafter referred to as “untreated seed potato”.
- Each treated and untreated seed meal was dried before germination.
- the seed amount per pot was about 2 g (about 70 grains) in terms of dry weight before the soaking process.
- Pots seeded with W3-treated seeds, W5-treated seeds and untreated seeds were designated as W3 treated group, W5 treated group and untreated group, respectively.
- the plants were cultivated in an artificial weather room set at 28 ° C. for 14 days, and the effects of the present invention were tested by comparing rice growth in each pot.
- the symptom evaluation was carried out using the following formulas to calculate the seedling ratio and control value of the rice seedling disease using the plant height height and the strain in which yellowing was observed as a seedling affected with rice seedling disease.
- Disease seedling rate (%) number of diseased seedlings / total number of standing seedlings x 100
- Control value (emergency seedling rate in untreated area-diseased seedling rate in treated area) / diseased seedling ratio in untreated area x 100
- the germination rate was approximately 90% or more with no significant difference in each treatment section.
- seedlings of seedling disease-resistant rice treated with Fusarium oxysporum non-pathogenic fungus W3 strain or W5 strain showed disease resistance against the rice seedling disease caused by another species Fusarium fujikuroi
- the seeds of seedling disease-resistant rice treated with microorganisms of biological control materials and settled are highly preventive and controllable against seedling diseases caused by pathogenic bacteria other than the biological control materials microbial species It was proved that
- Example 2 Effect of disease control in seedling disease-resistant plants (2) (the purpose) Preparation of microbial pesticides for controlling seedling diseases of the present invention, preparation of seeds by the method for producing seeds of disease-resistant plants using seedlings, and seedlings of the seeds are resistant to seedling diseases This was verified on a field scale.
- the host parent plant was rice (variety: short silver shaved) and cultivated by the following method in order to contact the non-pathogenic bacteria.
- the basic operation is in accordance with Example 1.
- seed sterilization of the parent rice was immersed in a disinfectant composed of a 200-fold diluted solution of 250 mg / L ipconazole / 230 mg / L cupric hydroxide wettable powder for 24 hours.
- the obtained sterilized parent rice seeds were bred according to a method (conventional method) known in the art.
- no control of rice sapling and seedling diseases was performed.
- rice (variety: Koshihikari) naturally infected with rice seedling disease was used as an infection source of rice seedling disease in the field.
- the seed pod of this infection source was raised without seed disinfection.
- the contact of the non-pathogenic fungus of the present invention with the parent rice is based on the heading period on the 88th day after the planting in which the heading rate is 40-50% and the heading period on the 91st day after the planting in which the heading rate is 80-90%.
- the non-pathogenic fungus was sprayed directly on the flower part using a hand spray during the morning (around 10 o'clock to 11 o'clock) when the parent rice bloomed twice.
- the spray amount was about 140 mL / m 2 .
- Parent rice after spraying was cultivated according to the customary method. On the 155th day after the planting of the parent rice, the rice was harvested, threshed, and seed pods were collected.
- the rice bud seedling disease incidence rate was verified for the various rice cakes obtained.
- the basic operation is in accordance with Example 1.
- the natural infection from Koshihikari which is infected with an illness and seedling disease in the field, is used as a contamination source, in principle, it is not necessary to inoculate the pathogen with a pathogen. Therefore, only the soaking treatment of the seed pod was tested for the disease inhibitory effect according to Example 1 using sterilized water instead of the rice sapling spore suspension.
- the disease symptom evaluation was carried out using the formula described in Example 1 to calculate the diseased seedling rate and the control value, with the plant height length and the strain in which yellowing was observed as a diseased seedling.
- the rate of seedling of rice sapling seedlings in seedling rice in the non-pathogenic fungus W3 treated area or W5 treated area seeded with seeds of disease resistant rice in the seedling stage was about 30% in the W3 treated area.
- the control value was 65% or more.
- the diseased seedling rate was only about 6.6%, and it had a control value of 90% or more.
- Example 3 Effect of disease control in seedling disease-resistant plants (3) It was verified by inoculating pathogenic bacteria under conditions more severe than those in Example 2 that seedling seedlings of the seedling stage disease-resistant plant seeds of the present invention were resistant to seedling stage diseases.
- Example 2 The materials and basic operations were the same as in Examples 1 and 2. However, in this example, as in Example 2, the non-pathogenic bacteria treatment is performed in the field, and the seed pods are prepared by using natural infection from the Kakahikari infection with the scab disease. Under the same conditions as in Example 1, it was immersed in a spore suspension of Fusarium fujikuroi, which was additionally inoculated with a pathogen. This is a control test that assumes that seeds contaminated with idiotic fungus remain in the seeds and that the seeds expand to adjacent strains during seedling raising.
- inoculation with rice sapling spore fungus spores in a suspension makes it possible to treat rice sapling in a non-pathogenic fungus W3 treatment area despite treatment under more severe disease conditions.
- the diseased seedling rate was about 20%, the control value was 62%, and even in the non-pathogenic fungus W5 treatment area, the rice seedling disease rate was about 15% and the control value was over 70%. Therefore, the effect of the present invention was demonstrated irrespective of the mildness of the infection conditions caused by the seedling pathogenic bacteria.
- Example 4 Effect of disease control in seedling disease-resistant plants (4) Even when a strain other than the Fusarium genus is used, it is confirmed that the seedling seedling seedling seedling seedling seedling seedling seedlings of the present invention are resistant to the seedling stage disease using a Trichoderma strain. It verified with.
- Example 1 Trichoderma atrovidide SKT-1 strain (trade name: Ecohope, Kumiai Chemistry) marketed as a microbial pesticide was used together with the non-pathogenic fungus W5 of Fusarium oxysporum.
- conidia were used by scraping those formed by plate culture instead of liquid culture. The concentration of conidia was 1.0 ⁇ 10 5 cells / mL, and spraying on the flower part was performed 4 times every 2 days from the 87th day after planting.
- the diseased seedling rate was only about 6.0%, and the control value was 50% or more. From this experiment, the effect of the present invention was demonstrated not only when the W3 strain and W5 strain, which are non-pathogenic bacteria of Fusarium oxysporum, but also when Trichoderma spp. Were used. Therefore, it is considered that the present invention can be practiced using a wide variety of bacteria including Fusarium and Trichoderma.
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Abstract
Description
本発明の第1の態様は、育苗期病害耐病性植物種子の製造方法(本明細書ではしばしば「製造方法」と称する)に関する。本発明の製造方法によれば、生物的防除資材微生物の利用により、育苗期病害に対してあたかも抵抗性品種のような耐病性を獲得した植物種子を製造し、それを提供することができる。
以下で本明細書において使用する各用語について定義する。
本明細書に記載の発明において使用する生物的防除資材微生物の分離方法について説明する。前述のように、生物的防除資材微生物は自然界に広く存在することから、植物体から比較的容易に分離することができる。例えば、Tateishi H. & Chida T. 2000, J. Gen. Plant Pathol., 66: 353-359に記載の方法を参照すればよい。
生物的防除資材微生物の分離源は、種子伝染性病害の発生圃場内においてその病害に罹患した植物株の周辺に生育する健全な同種株の植物組織(葉、胚軸、葉鞘、葉柄、茎、花器、果実、根部等)やその根圏土壌とすればよい。前記植物組織を分離源とする場合には、組織表面を消毒しておくことが好ましい。消毒は、例えば、採取した植物組織片約1cm2を70%エタノールに30秒間、続いて1%次亜塩素酸に3分間浸漬して組織表面を殺菌後、滅菌水で洗浄すればよい。ただし、エタノールや次亜塩素酸の濃度及び浸漬時間は特に限定するものでなく、植物組織の部位等によって当該分野で公知の技術に基づき、適宜調整すればよい。
生物的防除資材微生物が真菌の場合、表面殺菌後の植物組織を滅菌したピンセット、メス等を用いて1cm2程度の組織片に切り分けて培地上に置床する。培地は、PSA(ジャガイモ煎汁寒天培地:200 g/L ジャガイモ煎汁、0.5%[w/v]ショ糖、1.5%[w/v]寒天)やWA(素寒天培地:1% 寒天)を用いればよい。その他、分離すべき生物的防除資材微生物の属名が明確な場合には、その属に適合した選択培地を用いることもできる。細菌のコンタミネーションを防ぐため、培地上に予め20%乳酸を塗布しておくことが望ましい。前記植物組織を置床した培地を25~30℃で2~5日培養する。培地プレート上で生育した単一コロニー(単細胞真核微生物の場合)、菌糸先端(糸状菌又は担子菌の場合)、胞子又は分生子を新しい培地プレートに再移植して単一コロニー株を確立する。
分離した種子伝染性病原菌が非病原性菌、すなわち、本発明に使用する生物的防除資材微生物であるか否かは、上記(2)で単一株として確立した微生物を、宿主植物の苗に感染させ、その苗に特徴的な病害症状が現れないことを確認することによって分離すればよい。
本発明の育苗期病害耐病性植物の種子製造方法は、接触工程及び回収工程を含む。以下、各工程について具体的に説明をする。
「接触工程」とは、開花期前後の宿主植物の花部に生物的防除資材微生物を接触させる工程である。
「回収工程」とは、前記接触工程後に得られる前記生物的防除資材微生物が定着した宿主植物の種子を回収する工程である。前記接触工程で宿主植物の花部に接触した生物的防除資材微生物は、通常、頴娃や種皮や胚珠組織に定着する。宿主植物は、受粉後に非病原性変異株が定着した種子を発達させる。したがって、本工程では、発芽能力を有するまで十分に成熟した種子を当該分野で公知の方法により回収すればよい。接触工程後の宿主植物から得られる種子は、原則、育苗期病害耐病性植物の種子とみなすことができる。
本発明の育苗期病害耐病性植物の種子製造方法によれば、その種子の実生個体があたかも育苗期病害抵抗性品種のようにあらゆる育苗期病害に対して耐病性となる植物種子を簡便に製造し、提供することができる。
本発明の第2の態様は、育苗期病害の発病予防及び防除方法に関する。本発明によれば、育苗期病害耐病性植物種子の実生個体及び該実生個体周辺の他植物個体における育苗期病害の発病を予防し、また防除することができる。
本発明の育苗期病害の発病方法(以下、本明細書ではしばしば「予防防除方法」と称する)は、生物的防除資材微生物を定着させた第1態様に記載の育苗期病害耐病性植物の種子を用いる。本発明の予防防除方法の基本的工程は、育苗期病害耐病性植物の植物種における通常の栽培方法を行うだけでよい。ただし、播種に先立って行われる種子消毒については、行う必要はない。
本発明の予防防除方法によれば、種子伝染性病害や土壌伝染性病害を含む育苗期病害を、特別な処理を必要とすることなく、効率的に予防及び防除することができる。
本発明の第3の態様は、育苗期病害防除用微生物農薬に関する。本発明の育苗期病害防除用微生物農薬は、所望の植物に施用することで、その植物に育苗期病害耐病性を賦与することができる。
本発明の育苗期病害防除用微生物農薬は、生物的防除資材微生物を有効成分とする。
本発明の育苗期病害防除用微生物農薬によれば、育苗期病害耐病性植物を簡便に製造するための、又は宿主植物の感染を予防するための、安価で効率のよい剤を提供することができる。
育苗期病害耐病性植物における育苗期病害防除効果(1)
(目的)
本発明の育苗期病害防除用微生物農薬の調製、それを用いた育苗期病害耐病性植物種子の製造方法による種子の調製、及びその種子の実生個体が育苗期病害に対して耐病性となることをポットスケールで検証した。
生物的防除資材微生物には、Fusarium oxysporumの非病原性菌W3株(受託番号:NITE BP-01538)、及びW5株(受託番号:NITE BP-01539)を用いた。
発病苗率(%)=罹病苗数/全立苗数×100
防除価=(無処理区の発病苗率-処理区の発病苗率)/無処理区の発病苗率×100
育苗期病害耐病性植物における育苗期病害防除効果(2)
(目的)
本発明の育苗期病害防除用微生物農薬の調製、それを用いた育苗期病害耐病性植物の種子の製造方法による種子の調製、及びその種子の実生個体が育苗期病害に対して耐病性となることを圃場スケールで検証した。
Fusarium oxysporumの非病原性菌W5株(受託番号:NITE BP-01539)を用いた育苗期病害防除用微生物農薬の調製は、実施例1に準じて行った。培養液中の分生子の濃度は、1.0×105個/mLとした。
(目的)
育苗期病害耐病性植物における育苗期病害防除効果(3)
本発明の育苗期病害耐病性植物種子の実生個体が育苗期病害に対して耐病性となることを実施例2よりも厳しい条件で病原菌を接種して検証した。
材料及び基本操作は、実施例1及び2に準じた。ただし、本実施例では実施例2と同様に圃場で非病原性菌処理を行い、ばか苗病感染コシヒカリからの自然感染を汚染源として種籾を調製するものの、催芽処理において、種籾を滅菌水ではなく実施例1と同じ条件でイネばか苗病菌Fusarium fujikuroi胞子懸濁液に浸漬して、追加で病原菌接種を行った。これは、種子中にばか苗病菌の汚染したものが残存していて、育苗時に隣接株に拡大することを想定した防除試験である。
(目的)
育苗期病害耐病性植物における育苗期病害防除効果(4)
Fusarium属菌以外の菌株を用いた場合であっても、本発明の育苗期病害耐病性植物種子の実生個体が育苗期病害に対して耐病性となることを、Trichoderma属菌株を用いてポットスケールで検証した。
材料及び基本操作は、実施例1に準じた。ただし、本実施例では、Fusarium oxysporumの非病原性菌W5と共に、微生物農薬として市販されているTrichoderma atrovidide SKT-1菌株(商品名:エコホープ、クミアイ化学)を用いた。また、分生子は液体培養でなく、平板培養で形成されたものをかきとって使用した。分生子の濃度は1.0×105個/mLとし、花部への噴霧は、定植後87日目から、2日ごとに計4回行った。
Claims (15)
- 種子伝染性病原菌に対応する非病原性菌を開花期前後の宿主植物の花部に接触させる工程、
前記工程後に得られる前記非病原性菌が定着した宿主植物の種子を回収する工程
を含む、育苗期病害耐病性植物の種子製造方法。 - 前記宿主植物がイネ科植物である、請求項1に記載の製造方法。
- 前記イネ科植物がイネである、請求項2に記載の製造方法。
- 前記非病原性菌がFusarium属菌、Nectria属菌、Gibberella属菌、Calonectria属菌、Hypomyces属菌、Trichoderma属菌、Penicillium属菌、Talaromyces属菌、Acremonium属菌、Alternaria属菌、Verticillium属菌、Bacillus属細菌、Pseudomonas属細菌、Xanthomonas属細菌及びStreptomyces属細菌からなる群から選択される、請求項1~3のいずれか一項に記載の製造方法。
- 前記非病原性菌がFusarium oxysporum、Fusarium moniliforme、Fusarium fujikuroi、Fusarium proliferatum、及びFusarium sacchariからなる群から選択されるFusarium属菌由来である、請求項1~3のいずれか一項に記載の製造方法。
- 前記非病原性菌が受託番号NITE BP-01538又はNITE BP-01539である、請求項1~3のいずれか一項に記載の製造方法。
- 種子伝染性病原菌に対応する非病原性菌を開花期前後の宿主植物の花部に接触させることによって得られる育苗期病害耐病性植物の種子。
- 前記宿主植物がイネ科植物である、請求項7に記載の種子。
- 前記非病原性菌が請求項4~6のいずれかに記載の非病原性菌である、請求項7又は8に記載の種子。
- 請求項7又は8に記載の育苗期病害耐病性植物の種子を用いる育苗期病害の発病予防及び防除方法。
- 前記植物がイネ科植物である、請求項10に記載の予防及び防除方法。
- 前記育苗期病害が種子伝染性病害及び土壌伝染性病害である、請求項10又は11に記載の予防及び防除方法。
- 種子伝染性病原菌に対応する非病原性菌を有効成分とする育苗期病害防除用微生物農薬。
- イネ科植物適用用である、請求項13に記載の微生物農薬。
- 前記非病原性菌が請求項4~6のいずれかに記載の非病原性菌である、請求項13又は14に記載の微生物農薬。
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CN114902904A (zh) * | 2022-06-20 | 2022-08-16 | 江西省永盛园艺股份有限公司 | 一种多肉植物养育用环境消杀设备 |
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JP2016123372A (ja) * | 2015-01-06 | 2016-07-11 | 日本車輌製造株式会社 | 自動種子消毒装置 |
US10412682B2 (en) * | 2017-08-30 | 2019-09-10 | Qualcomm Incorporated | Mechanism to update/download profile using low power or no power |
CN114456955B (zh) * | 2022-02-25 | 2024-03-19 | 中国水稻研究所 | 一种水稻恶苗病的高效接种方法及其应用 |
CN115466685B (zh) * | 2022-08-11 | 2024-02-23 | 浙江钱江生物化学股份有限公司 | 藤仓镰刀菌及其发酵生产赤霉素a4+7的方法及运用 |
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JP2001346407A (ja) * | 2000-06-08 | 2001-12-18 | Ts Shokubutsu Kenkyusho:Kk | 無病種子の生産方法 |
JP2003192515A (ja) * | 2001-12-27 | 2003-07-09 | Kureha Chem Ind Co Ltd | 植物病害防除剤および防除方法 |
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JP2001346407A (ja) * | 2000-06-08 | 2001-12-18 | Ts Shokubutsu Kenkyusho:Kk | 無病種子の生産方法 |
JP2003192515A (ja) * | 2001-12-27 | 2003-07-09 | Kureha Chem Ind Co Ltd | 植物病害防除剤および防除方法 |
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CN114902904A (zh) * | 2022-06-20 | 2022-08-16 | 江西省永盛园艺股份有限公司 | 一种多肉植物养育用环境消杀设备 |
CN114902904B (zh) * | 2022-06-20 | 2023-07-25 | 江西省永盛园艺股份有限公司 | 一种多肉植物养育用环境消杀设备 |
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JP2014195451A (ja) | 2014-10-16 |
KR20150128883A (ko) | 2015-11-18 |
US10264796B2 (en) | 2019-04-23 |
JP6241001B2 (ja) | 2017-12-06 |
KR101770656B1 (ko) | 2017-08-23 |
US20160015040A1 (en) | 2016-01-21 |
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