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WO2023191365A1 - Souche d'irpex lacteus am003, effet de lutte contre la maladie des cultures provoquée par celle-ci, et applications agro-industrielles - Google Patents

Souche d'irpex lacteus am003, effet de lutte contre la maladie des cultures provoquée par celle-ci, et applications agro-industrielles Download PDF

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WO2023191365A1
WO2023191365A1 PCT/KR2023/003692 KR2023003692W WO2023191365A1 WO 2023191365 A1 WO2023191365 A1 WO 2023191365A1 KR 2023003692 W KR2023003692 W KR 2023003692W WO 2023191365 A1 WO2023191365 A1 WO 2023191365A1
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strain
plant
spores
culture
sclerotia
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이성기
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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi

Definitions

  • the present invention relates to Ilpex lateus strains having plant pest prevention effects and their uses.
  • Plant anthracnose affects over 300 types of crops and plants, including peppers, persimmons, apples, pears, grapes, ginseng, rice, Schisandra chinensis, beans, watermelons, strawberries, cucumbers, chestnuts, plums, Asteraceae plants, vegetables, trees, flowers, and coffee trees. It is mainly caused by the genera Glomerella and Colletotrichum , and the damage is very serious.
  • Pepper anthracnose is one of the major inhibitors of pepper production along with pepper blight.
  • Five types of anthracnose cause disease in leaves, stems, branches, seeds, and fruits.
  • the damage caused by fruit anthracnose, which mainly occurs on mature fruits, is more than 95%.
  • the damage is most direct and fatal.
  • Anthracnose has recently become a major problem in pepper cultivation.
  • pepper anthracnose is caused by the genus Colletotrichum , which belongs to the Ascomycota phylum of the fungi kingdom. In the seedling stage, it is Colletotrichum coccodes , and in pepper fruits, it is Colletotrichum cloeos. It is caused by C. gloeosporioides and C. acutatum . This pathogen forms ascospores and conidia, spreads them to the surrounding area, and overwinters in the form of hyphae or conidia.
  • the optimal temperature for the onset of the disease is 26-28°C, and it is easy to develop the disease in a humid environment.
  • Anthracnose mainly occurs in green peppers from the time they are produced until harvest time.
  • Plant anthracnose as described above has been achieved through the treatment of numerous pesticides.
  • chemical pesticides has resulted in side effects such as pesticide poisoning, groundwater contamination, soil contamination, toxicity to humans and livestock, residues in agricultural products, and emergence of resistant organisms to various pests and weeds due to indiscriminate abuse.
  • the Green Round an international trade multilateral negotiation linking the environment and trade, was in full swing, providing food to humanity. Farming also needed to be converted to environmentally friendly agriculture.
  • biological pesticides that use microorganisms themselves or their functions directly and indirectly have recently been attracting attention.
  • Bio pesticides are defined as commercialized products of microorganisms, natural substances, natural enemies, etc. collected separately from the natural environment to control crop pests, pathogenic microorganisms, and weeds. These biological pesticides have the advantage of having a longer onset and duration of efficacy compared to chemical pesticides, the development period and development cost of pesticides are cheaper than chemical pesticides, and there are no side effects such as toxicity to humans and livestock and environmental destruction.
  • Pests and pests occur at the intersection of a variety's disease resistance, environment, and pathogens under special conditions, and in this case, the role of a balancer is important. Pathogen transmission through seeds, air, soil, and insects is a process that can be controlled by the action of microbial balancers.
  • the present inventor seeks to provide the following novel microorganisms and their uses.
  • One aspect of the present invention is to provide a microbial pesticide preparation suitable for use as white rot fungi of the white rot fungus class.
  • the aim is to provide a balancer microorganism that can control the homeostasis of the ecosystem biosphere, and the present invention isolates and identifies a new technological keystone species strain from the natural world to prevent the occurrence of plant diseases, including plant anthracnose.
  • one aspect of the present invention is anthracnose, which causes damage to over 900 types of plants worldwide, and also occurs in Korea, like anthracnose, in most crops such as apples, peppers, tomatoes, garlic, onions, lettuce, and rice.
  • the purpose is to provide an Irpex lacteus am003 strain capable of controlling anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice leaf sheath blight, and microbial preparations containing the same.
  • one aspect of the present invention is a microorganism capable of controlling any one or more of apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and/or rice sheath blight. We would like to provide a formulation.
  • one aspect of the present invention seeks to provide a technological keystone species strain that can solve these difficult problems in a natural enemy manner and become a balance regulator of the ecosystem.
  • the purpose of the present invention is to provide a strain of Irpex lacteus am003 ( Irpex lacteus am003) deposited with accession number KACC 83059 BP.
  • Another object of the present invention is the Ilpex Latheus am003 strain; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; and extracts of the strains, lysates, cultures or fermentations;
  • the aim is to provide a method for controlling apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice sheath blight, including the step of using one or more of the following.
  • Another object of the present invention is the Ilpex lateus am003 strain; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; and extracts of the strains, lysates, cultures or fermentations;
  • the object is to provide a method for controlling plant pathogenic viruses, including the step of using one or more of the following.
  • Another object of the present invention is the Ilpex lateus am003 strain; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; and extracts of the strains, lysates, cultures or fermentations; It is intended to improve the soil strength of farmland by using a soil purification and restoration method that includes the step of using one or more of the following.
  • One aspect of the present invention provides a strain of Irpex lacteus am003 ( Irpex lacteus am003), which has antibacterial activity against nine types of crop disease control and plant pathogenic viruses.
  • strains according to mycological taxonomy are Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricomycetes (Class Agaricomycetes), Polyporales (Order Agaris), Meruliaceae (Glyaceae), and Irpex (Irpex).
  • Mushroom genus a strain of white rot fungi of the I. lacteus species. It is a new wild species isolated from a mountainous forest area in Gyeonggi-do. The white rot fungi strain decomposes lignin, produces medicinal substances, and produces harmful toxic substances. Its biodegradation and soil improvement effects are already well known.
  • Strains according to one aspect of the present invention are crop anthrax Glomerella sp ., Colletotrichum sp . and apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice sheath stain that occur in most crops such as peppers, tomatoes, apples, garlic, onions, lettuce, and rice.
  • This is a technology related to the Irpex lacteus am003 strain that can control blight and has excellent control effects on 9 types of diseases.
  • the strain provides a strain that is a highly heat-resistant strain capable of surviving at a temperature of 110°C or more and for a time of more than 30 minutes.
  • the plant anthrax is provided as a strain selected from the genus Glomerella and Colletotrichum genus Anthrax, and 9 types of disease pathogens.
  • the strain includes the DNA barcode marker base sequence of ITS 1 and 2 of the rDNA structure (nuclear ribosomal RNA cistron).
  • the strain is Irpex lacteus am003 ( Irpex lacteus am003).
  • the strain provides a strain whose accession number is Irpex lacteus am003 (KACC 83059BP).
  • Irpex lacteus am003 is capable of controlling Glomerella , Colletotrichum anthrax, apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot sclerotia, lettuce sclerotia, basil sclerotia, gray mold, and rice leaf sheath blight. As a technology related to strains, it has excellent control effects on 9 types of plant diseases.
  • Another aspect of the present invention is a strain according to any one of the above; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • a microbial preparation comprising one or more of the following.
  • Another aspect of the present invention is a strain according to any one of the above; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides nine types of disease control methods, including plant anthracnose, including the step of using one or more of the following.
  • the plants include pepper, lettuce, rice, persimmon, apple, pear, grape, ginseng, deodeok, bellflower root, barley, wheat, corn, Schisandra chinensis, goji berry, blueberry, talisman, jujube, plum, soybean, Peanuts, potatoes, sweet potatoes, watermelons, melons, tomatoes, strawberries, pumpkins, cucumbers, chestnuts, peaches, plums, tangerines, bananas, pineapples, mangoes, olives, pomegranates, cherry, dragon fruit, walnuts, garlic, onions, radishes, cabbage, One or more of plum, asteraceae plants, orchids, flowers, coffee trees, pine trees, pine trees, and other vegetables and trees, plant anthracnose, apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, earthy rot sclerotia, lettuce sclerotia, basil sclerotia, This is a technology related
  • Another aspect of the present invention is a strain according to any one of the above; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • nine types of disease control methods including plant anthracnose, using one or more of the following, it contributes to improving agricultural soil strength as a soil purification and restoration method.
  • strains according to mycological taxonomy are Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricomycetes (Class Agaricomycetes), Polyporales (Order Agaris), Meruliaceae (Glyaceae), and Irpex (Irpex).
  • Mushroom genus a strain of white rot fungi of the I. lacteus species. It is a new wild species isolated from a mountainous forest area in Gyeonggi-do. The white rot fungi strain decomposes lignin, produces medicinal substances, and produces harmful toxic substances. Biodegradation and soil improvement are already well known.
  • crop anthrax Glomerella sp. and Colletotrichum sp. It has an excellent control effect against pathogens such as apple anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice sheath blight.
  • Strains according to one aspect of the present invention can be easily cultured in laboratories and factories at 25°C on PDA (potato dextrose agar, pda) and PDB (potato dextrose broth, pdb) media.
  • the strain according to one aspect of the present invention is capable of early large-scale production, and is effective against diseases such as anthracnose, late blight, wilt, sclerotia, gray mold, and sheath blight of agricultural crops such as apples, peppers, garlic, onions, lettuce, and rice. It can be applied in various ways.
  • the strain according to one aspect of the present invention can make an innovative contribution to the cultivation of eco-friendly and organic crops as a dedicated microbial agent. It can be an excellent product for registering natural pesticides as a pure microbial preparation and improving technological eco-friendly agriculture, significantly ahead of domestic and foreign bio preparations.
  • the strain according to one aspect of the present invention has high heat resistance and is suitable for fertilization between early March and October depending on the pepper and each farming season. This can result in a more efficient function when temperatures are high even during different crop seasons.
  • the strain according to one aspect of the present invention is a description of the Irpex lacteus am003 strain capable of controlling apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot sclerotia, lettuce sclerotia, basil sclerotia, gray mold, and rice leaf sheath blight.
  • it shows the selective metabolism of the molecular signaling process that decomposes and kills the hyphae of the infectious agent and uses them as a carbon source, as well as the ventral growth of hyphae that selectively controls the infection site of 9 types of pathogens.
  • Figure 1 is a photograph of the strain isolated from a mountainous area in Gyeonggi-do, Korea in Example 1 of the present invention after 2 weeks and 1 week of pure subculture in PDA medium.
  • Figure 2 is a photograph of pure isolation of the strain of the present invention after 1 week of pdb liquid culture.
  • Figure 3 is a diagram of the ITS sequence of the am003 strain.
  • Figures 4a and 4b are the results of ITS sequence analysis of each colony (same medium) of the Irpex lacteus am003 strain of the present invention.
  • Figure 4c is a schematic diagram of the Irpex lacteus am003 strain of the present invention.
  • Figure 5 is a photograph showing the control of each pathogen by the Irpex lacteus am003 strain on pda medium. Inhibition and death of pathogens and ventral growth of am003 during re-cultivation by inoculating the pathogen (middle spot) and the am003 strain (both sides). form (white part hyphae) (5a to 5e).
  • Figure 5a shows the results of the mutual reaction between pathogens of pepper blight, gray mold, and lettuce sclerotia and the Irpex lacteus am003 strain.
  • Figure 5b is the result of the mutual reaction between the pathogens of pepper blight and pepper wilt disease and the Irpex lacteus am003 strain.
  • Figure 5c shows the results of the mutual reaction between pathogens of rice leaf sheath blight, basil sclerotia, and lettuce sclerotia and the Irpex lacteus am003 strain.
  • Figure 5d shows the results of the mutual reaction between the pathogens of apple anthracnose and soil rot sclerosis and the Irpex lacteus am003 strain.
  • Figure 5e shows the results of the mutual reaction between pathogens of basil sclerotia, gray mold, apple anthracnose, and rice sheath blight and the Irpex lacteus am003 strain.
  • Figure 5f is the result of confirming whether pathogenicity of the am003 strain occurred in pepper seedlings after inoculation of the Irpex lacteus am003 strain into pepper pot seedlings, and it was confirmed that the seedlings grew more robustly and were not pathogenic.
  • Figure 5g is the result of checking the inside of the stem epidermis of the pepper plant to see whether pathogenicity of the am003 strain occurred in pepper seedlings after inoculation of the Irpex lacteus am003 strain into pepper pot seedlings. It was confirmed that the seedlings grew more robustly and were not pathogenic. did.
  • Figure 6 is a photograph summarizing the pesticide-free clean pepper production process using the am003 culture strain in an open field pepper field test plot.
  • Figure 6a shows the cultured strain stock solution of Irpex lacteus am003.
  • Figure 6b is a photograph of the blending operation for homogenization of the mycelia of the am003 stock culture strain.
  • Figure 6c shows the 200- to 400-fold dilution process of the homogenized am003 cultured strain.
  • Figure 6d is a photograph of the homogenized and diluted am003 cultured strain prepared for spraying in an open field pepper field.
  • Figure 6e is a photograph of the spraying process of am003 diluted cultured strain in an open field pepper field.
  • Figure 6f shows the production of pesticide-free clean peppers from open field pepper fields by regular (10 to 20 days) administration of the am003 strain.
  • the variable when a range is stated for a variable, the variable will be understood to include all values within the stated range, including the stated endpoints of the range.
  • the range “5 to 10” includes the values 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood that it also includes any values between integers that fall within the scope of the stated range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9, etc.
  • the range “10% to 30%” includes the values 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, as well as 10% to 15%, 12%, etc. It will be understood that it includes any subranges, such as from 18% to 18%, from 20% to 30%, etc., and also includes any values between reasonable integers within the range of the stated range, such as 10.5%, 15.5%, 25.5%, etc.
  • the strain of the present invention is the Irpex lacteus am003 strain isolated from a mountainous forest area in Korea, and the strain was confirmed to have the effect of improving soil strength and selectively controlling and preventing anthrax in open pepper field test plots during the pepper production process.
  • the strain of the present invention is a technology related to the Irpex lacteus am003 strain, which can control apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice leaf sheath blight, It shows the metabolic process of molecular signaling that recycles the hyphae as a carbon source while decomposing and killing the hyphae of the infectious agent and the ventral growth of hyphae that selects and controls the infection site of 9 types of pathogens.
  • the strain of the present invention was confirmed to be a highly heat-resistant strain that survives sterilization at 121 degrees Celsius for 30 minutes without dying, and such function is believed to efficiently control 9 types of diseases, including anthracnose, in the high-temperature maturation environment of crops.
  • zoospores swim along the water to reach the host and invade the plant.
  • zoospores lose their swimming hairs, become thickened, and different types of thickened spores combine to form round-shaped oospores.
  • Plague bacteria overwinter in the soil in the form of oospores and become a source of infection the following year.
  • the oospores have very strong resistance to the surrounding environment and can survive in the soil for 2 to 2 years. Pathogens grow well in humid places and slightly acidic soils because of their inherent connection with water.
  • Red pepper blight bacteria develop quickly in high temperature and high humidity environments, causing a high bacterial load, and in dry and well-ventilated situations, the survival rate is relatively low and the entire pathogen dies after 2 to 4 days.
  • Apple anthrax pathogen Glomerella cingulata
  • Conidia are colorless single cells, oval or cylindrical, and when wet, they turn light red and leak as mucus.
  • the asci are black, spherical to flask-shaped, and the asci within them are club-shaped.
  • Anthracnose mainly occurs on fruit, but sometimes also occurs on tree branches and tree trunks.
  • Pepper anthracnose fungi include Colletotrichum acutatum , C. cocodes , C. dematium , C. gloeosporioides , etc.
  • C. acutatum is widespread and causes anthracnose not only in peppers but also in major fruit trees such as apples, pears, grapes, persimmons, and peaches, as well as many other crops.
  • the optimal temperature for growth is around 26-30°C, and it overwinters in diseased fruits discarded last year. If the size of the lesion formed on the fruit is more than 1.0 cm, the number of conidia, which are secondary infectious agents, is tens of millions or more, and in some cases, more than 100 million are formed in one lesion.
  • Fusarium oxysporum is a fungus belonging to the order Fusarium oxysporum .
  • Pathogens form large amounts of small spores (egg or kidney-shaped, mainly single-celled), large-sized spores (sickle-shaped, 3-celled), and endospores called chlamydospores. It grows well in a culture medium (PDA), forming many white aerial hyphae. As the colony ages, a purple pigment is formed, and areas where many sclerotia are formed may take on a dark ink color. It forms large amounts of spores inside plant cell tissues and outside diseased tissues.
  • PDA culture medium
  • the occurrence and damage of this disease vary greatly depending on the density of pathogens, weather conditions during the growth period, especially temperature and humidity, fertilization management, and planting methods.
  • the temperature at which pathogens can invade rice tissue is 22 to 35°C, and the optimal temperature is 30 to 32°C, and outbreaks increase if the humidity between plants continues to be above 96%.
  • Gray mold is a disease that is mainly a problem in house green pepper cultivation. When growing seedlings in cold beds for outdoor cultivation, it may occur in young seedlings in the early stages of the seedling period. Damage occurs in the form of flower and fruit rot and stem dieback. It is caused by invasion of a fungus belonging to the imperfect fungus called Botrytis cinerea . In the lesion, brown conidia grow erect and dense, and the ends of the conidia are slightly swollen and have small protrusions.
  • the pathogen spends the winter in the form of sclerotia in the tissues and soil of the diseased plant or in the mycelial state within the infected plant, then germinates to form ascospores and ascospores.
  • Ascospores attach to and invade weak parts of the plant, and hyphae that germinate and extend from the sclerotia and mycelium may directly invade the plant.
  • the pathogen Sclerotinia minor spends the winter in the form of sclerotia in the tissues and soil of diseased plants or in the form of hyphae in the infected plant, then germinates to form ascospores and ascospores.
  • ITS internal transcribed spacer
  • Basil Ocimum basilicum
  • Ocimum basilicum is an annual herb that is native to Africa and tropical Asia and is grown all over the world, including Korea and Europe. It is mainly sown in April to May and transplanted in June (Groom, 1992). The leaves and stems are dried and used as a cooking spice, and the aromatic oil is used to scent beverages, cosmetics, perfume, toothpaste, and soap (Karawya et al., 1974). Taxonomically, it is Ocimum basilicum .
  • Black rot sclerotia is a problem in field garlic cultivation areas where garlic is continuously grown, and the pathogen ( Sclerotium cepivorum ) is a type of soil-borne pathogen that forms black, spherical or oblate sclerotia.
  • the size of sclerotia is usually 0.5 to 0.6 mm, which is much smaller than the sclerotia of other sclerotia diseases. Its host range is to invade plants of the Allium genus, including green onions, onions, and chives. It also occurs in onion cultivation areas in the southern region of Korea, causing great damage.
  • the pathogens of the above crop diseases are mostly molds and fungi, which are microbial species that are extremely difficult to control with pesticides and biological agents.
  • the present invention discovers new strains of Keystone species that can become a balancer of the ecosystem and provides fundamental solutions. We want to establish effective measures. We seek to discover microbial strains in the natural world that can function as balancers of agricultural and natural ecosystems and utilize them in accordance with ecological principles. It will be able to play a role in improving clean organic agriculture and restoring the global environment.
  • the present invention is an Irpex lacteus am003 strain, which has an innovative disease control function that can control apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice sheath blight.
  • Irpex lacteus am003 of the present invention is suitable for most agricultural crops other than pepper (persimmon, apple, pear, grape, ginseng, deodeok, bellflower, rice, barley, wheat, corn, Schisandra chinensis, goji berry, blueberry, talisman, jujube, plum, soybean, peanut) , potato, sweet potato, watermelon, melon, tomato, strawberry, pumpkin, cucumber, chestnut, peach, plum, tangerine, banana, pineapple, mango, olive, pomegranate, cherry, dragon fruit, walnut, garlic, onion, lettuce, radish, Chinese cabbage.
  • pepper persimmon, apple, pear, grape, ginseng, deodeok, bellflower, rice, barley, wheat, corn, Schisandra chinensis, goji berry, blueberry, talisman, jujube, plum, soybean, peanut
  • potato sweet potato, watermelon, melon, tomato, strawberry, pumpkin,
  • plum asteraceae plants, orchids, flowers, coffee trees, pine trees, pine trees, other vegetables, trees, etc.), apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot sclerotia, lettuce sclerotia, basil sclerotia, gray mold. It is a microbial agent that can be used to treat diseases such as rice leaf sheath blight.
  • the present invention is a method for controlling and controlling nine types of disease infections, such as anthracnose of peppers and other crops, using culture medium or sterilized (121°C) culture medium of the strain of the present invention cultured in PDB (potato dextrose broth) medium and other composition media. to provide.
  • culture medium or sterilized (121°C) culture medium of the strain of the present invention cultured in PDB (potato dextrose broth) medium and other composition media. to provide.
  • the present invention relates to apple anthracnose, pepper anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice leaf sheath using live bacterial cultures of the strain of the present invention cultured in PDB medium and other prepared media. Provides a method to control and control diseases such as spot blight.
  • the present invention involves centrifuging the culture medium live cells of Irpex lacteus am003, which is the strain of the present invention, the culture liquid sterilized body, and the culture medium live bacteria, followed by filtration using various filtration methods such as pure supernatant and membrane, and then pure filtrate and these filtrate and supernatant. , provides methods and products used for pest control of 9 types of diseases, soil improvement, and soil improvement in the form of powders, granules, and homogenized liquid culture diluents made by reprocessing pure culture fluids.
  • This invention has the deposit number KACC 83059
  • KACC 83059 We provide a new wild isolated strain of BP, Irpex lacteus am003, and microbial preparations containing it.
  • the present invention is directed to larvae control of flower yellow thrips vectors in grass fields such as chickweed around pepper fields, which includes the step of pre-spraying Irpex lacteus am003, which is a strain of the present invention, and a microbial agent containing it (due to the characteristics of am003, larval penetration mycelial function and It provides a fundamental control method for TSWV infection by killing larvae through mycelial proliferation.
  • the present invention provides a method for preventing nine types of crop diseases, comprising the step of spraying Irpex lacteus am003, a strain of the present invention, and a microbial preparation containing it to any one or more of the soil and moisture supplied to the target plant.
  • the present invention provides a method for controlling larvae of insects, moths, etc. of other crops, including pine wilt nematode, comprising the step of spraying Irpex lacteus am003, which is the strain of the present invention, and a microbial agent containing it.
  • One aspect of the present invention provides an Irpex lacteus strain that has antibacterial activity against plant pathogenic microorganisms and plant pathogenic viruses.
  • strains according to mycological taxonomy are Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricomycetes (Class Agaricomycetes), Polyporales (Order Agaris), Meruliaceae (Glyaceae), and Irpex (Irpex).
  • Mushroom genus a strain of the I. lacteus species, is a new wild species purely isolated from the mountainous forest area of Gyeonggi-do, and is an agricultural anthrax, Glomerella , and Colletotrichum sp. It has an excellent control effect against pathogens such as apple anthracnose, pepper blight, pepper wilt, soil rot, lettuce sclerotia, basil sclerotia, gray mold, and rice sheath blight.
  • the strain provides a strain that is a highly heat-resistant strain capable of surviving at a temperature of 110°C or more and for a time of more than 30 minutes.
  • the plant pathogenic microorganism is any one or more of plant anthrax, plant blight, plant wilt-causing bacteria, plant sclerotia-causing bacteria, plant fungal disease-causing bacteria, and sheath blight-causing bacteria, and provides a strain. .
  • the plant pathogenic virus is a tomato spotted wilt virus (TSWV) strain.
  • TSWV tomato spotted wilt virus
  • the plant anthrax is anthrax of the genus Glomerella and It is one or more selected from anthrax of the genus Colletotrichum
  • the plant blight bacteria are a genus of Phytophotora
  • the bacteria causing plant wilt are a bacterium of the genus Fusarium
  • the bacteria causing plant sclerotia are The bacterium is a genus of Scerotinia
  • the plant fungal disease-causing bacterium is a genus of Botrytis
  • the bacterium causing leaf sheath blight is a genus of Thanatephorus .
  • the strain includes the DNA barcode marker base sequences of ITS 1 and 2 of the rDNA structure (nuclear ribosomal RNA cistron) of Sequence Listing 1.
  • the strain is Ilpex Latheus am003.
  • the strain provides a strain whose accession number is KACC 83059BP.
  • Another aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; Provided is a microbial preparation comprising one or more of the following.
  • the second aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling plant anthracnose, comprising: using one or more of the following.
  • the plants include pepper, persimmon, apple, pear, grape, ginseng, deodeok, bellflower root, rice, barley, wheat, corn, Schisandra chinensis, goji berry, blueberry, blueberry, jujube, plum, soybean, Peanuts, potatoes, sweet potatoes, watermelons, melons, tomatoes, strawberries, pumpkins, cucumbers, chestnuts, peaches, plums, tangerines, bananas, pineapples, mangoes, olives, pomegranates, cherry, dragon fruit, walnuts, garlic, onions, lettuce, radish,
  • Provided is a method for controlling plant anthracnose in one or more of cabbage, plum, Asteraceae plants, orchids, flowering plants, coffee trees, pine trees, and pine trees.
  • the second aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • a composition for controlling plant anthracnose comprising one or more of the following.
  • the third aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling plant blight comprising: using one or more of the following.
  • the plant blight is caused by bacteria of the genus Phytophotora , and the plant blight is pepper blight, providing a method for controlling plant blight.
  • the third aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a composition for controlling plant blight, comprising one or more of the following.
  • the fourth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling plant wilt disease comprising: using one or more of the following.
  • the plant wilt disease is caused by fungi of the genus Fusarium , and the plant wilt disease is pepper wilt disease, providing a method for controlling plant wilt disease.
  • the fourth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • a composition for controlling plant wilt disease comprising one or more of the following.
  • the fifth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling plant sclerotia disease comprising: using one or more of the following.
  • the plant sclerotia disease is caused by fungi of the genus Scerotinia , and the plant sclerotia disease is any one or more of soil rot sclerotia, lettuce sclerotia, and basil sclerotia, a method for controlling plant sclerotia to provide.
  • the fifth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a composition for controlling plant sclerotia diseases, comprising one or more of the following.
  • the sixth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling plant fungal diseases comprising: using one or more of the following.
  • the plant fungal disease is caused by bacteria of the genus Botrytis , and the plant fungal disease is gray mold disease, providing a method for controlling plant fungal diseases.
  • the sixth aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • a composition for controlling plant fungal diseases comprising one or more of the following.
  • the seventh aspect of the present invention is a strain in any one of the aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products; It provides a method for controlling leaf sheath blight including the step of using one or more of the following.
  • the sheath blight is caused by bacteria of the genus Thanatephorus , and the sheath blight is a rice sheath blight, providing a method for controlling sheath blight.
  • the seventh aspect of the present invention is a strain according to any one of the above aspects of the present invention; its spores; Its hyphae; its shredded material; its culture; Fermented products thereof; And extracts of the above strains, spores, mycelia, lysates, culture broth or fermentation products;
  • a composition for controlling leaf sheath blight comprising one or more of the following.
  • Basidiomycetes belonging to the class Mushrooms are useful fungi of the Basidiomycete family that are of rare value as their species and ecological functions are closer to human medicine and soil restoration than other fungi, and are more easily identified morphologically. . While exploring these basidiomycetes, it was thought that their functions were not only for simple environmental purification and medicinal purposes, but also for metabolically controlling pathogens that cause pests and diseases, and for their ecological and evolutionary connection. In most cases, new wild species are composed of complex systems as symbionts of numerous biological species, like the human body or a mixture of gemstones. Therefore, in most cases, it is very difficult to isolate the pure specific species.
  • mycelia and spores were separated from the fruiting body form of white rot fungi at the spore and mycelium level on a specific medium such as PDA, more than 200 times over a period of one year. Afterwards, the starting point of mycelial spores that appeared morphologically pure was identified, and their purity was secured by subculturing them more than 100 times.
  • Figure 1 is a photograph of an isolated strain after culturing it in PDA medium for 1 to 2 weeks.
  • Example 1 The strain isolated in Example 1 had septum spores and white, long, spiral, rod-shaped hyphae. Looking at Figure 2, you can see a photo of the strain of Example 1 cultured in PDB medium for 1 to 2 weeks and a photo of the mycelium's tissue. At the beginning of culture, pda fragments form thin hyphae over 2 to 4 days, and after 7 days, white lump-shaped circular hyphal protrusions of thick mycelial complexes are formed for a long period of time.
  • This morphological change is thought to spatially control the activity of the pathogen while the antibacterial antagonists produced by the cells of the strain isolated in Example 1 block the function of the pathogen at its source.
  • the hyphae as described above have septum cells, and it is understood that the septum cells perform a signal transduction process that detects nine types of pathogens such as plant blight and anthrax and the function of secreting control antagonists.
  • Example 3 Identification of am003 strain (strain identification and classification based on ribosomal RNA operon, a genomic marker)
  • Example 2 As a result of identifying the strain isolated in Example 1 and whose physiological and morphological characteristics were identified in Example 2, it was confirmed that it had a white rot fungal strain of Irpex lacteus belonging to the class of Mushrooms. Specifically, the following process was followed.
  • the plate culture medium strain was submitted to Mokwon University's Institute of Microbial Ecological Resources (IMER) for analysis. Strain identification by analysis of ITS rRNA sequence was conducted at Mokwon University's Institute of Microbial Ecological Resources (IMER).
  • the PCR purified product was analyzed by comparing the ITS 1 region nucleotide sequence of approximately 558 to 609 bp using Genetic Analyzer 3730 (Applied Biosystems).
  • the homology of the ITS region gene sequence of the effective microorganism am003 was confirmed using SeqMan software (DNAStar) and the BioEdit program, and the phylogenetic tree was created using the MEGA 5.2 program.
  • ITS sequence is a universal DNA barcode marker for classifying and identifying fungal strains. Eukaryotic fungi do not undergo 16S rRNA analysis as do common bacterial strains. The rRNA commonly contained in eukaryotic fungi includes 5.8S ribosomal RNA and large subunit (26, 28S) ribosomal RNA, and ITS (internal transcribed spacer) 1, which is the 18 S RNA sequence of a specific region of these rRNAs. 2 are being analyzed, and among them, ITS 1 region was compared. This is the same as Figure 3.
  • ITS sequence analysis DNA was extracted from the fungal culture of Example 1, and a universal primer capable of tracking the ITS sequence was amplified by PCR to determine the DNA sequence (PCR amplification ⁇ electrophoresis ⁇ barcode sequencing ⁇ sequence editing ⁇ Barcode library). Afterwards, the sequence was grafted onto the NCBI data base and classified and identified by comparison of homology. The sequence appears as DNA, but its origin is ribosomal RNA. In other words, identification is performed using ITS1 and 2 as universal barcode markers.
  • ITS rDNA DNA sequence that code for ribosomal RNA
  • Universal primer PCR nucleotide
  • the DNA sequence appears and displays the sequence. This is the sequence below.
  • the ITS 1 sequence of the randomly selected am003 strain was analyzed and compared to the above sequence list 1 and two mycelial colonies of the am003 strain on the same pda plate medium for pure identity testing.
  • the isolated strain was confirmed to be a strain of Irpex lacteus and was named am003.
  • the phylogenetic diagram of the isolated strain was shown in Figure 4c.
  • am003 is effective against 9 plant diseases (apple, pepper anthracnose, pepper blight, pepper wilt, gray mold, lettuce sclerotia, basil sclerotia, soil rot sclerotia, and rice sheath blight).
  • the experimental method was initial liquid culture of the standard pathogen and the am003 strain from the Chungnam Agricultural Research and Extension Services (a regional research institute of the Rural Development Administration). Then, after culturing the pda solid medium, each section of the solid medium was taken at a size of 1 to 2 mm (inoculated with the number of viable pathogens approximately 5 to 10 times higher than that of the control strain), and inoculated into the center of the new pda solid medium ( pathogen) and both sides (am003). Changes in reaction were observed while culturing in pda medium in an incubator at 25°C for 3 to 14 days.
  • Figure 5a shows the results of the mutual reaction between pathogens of pepper blight, gray mold, and lettuce sclerotia and the Irpex lacteus am003 strain.
  • Figure 5b is the result of the mutual reaction between the pathogens of pepper blight and pepper wilt disease and the Irpex lacteus am003 strain.
  • Figure 5c shows the results of the mutual reaction between pathogens of rice leaf sheath blight, basil sclerotia, and lettuce sclerotia and the Irpex lacteus am003 strain.
  • Figure 5d shows the results of the mutual reaction between the pathogens of apple anthracnose and soil rot sclerosis and the Irpex lacteus am003 strain.
  • Figure 5e shows the results of the mutual reaction between pathogens of basil sclerotia, gray mold, apple anthracnose, and rice sheath blight and the Irpex lacteus am003 strain.
  • the mutual reaction between the standard strain of the pathogen and the am003 strain involves the gradual suppression or death of the standard strain of the Agricultural Research and Extension Services pathogen as the white mycelia of am003 overgrow the standard strain over a period of 3 to 14 days.
  • the phenomenon of am003 being used as a carbon source was commonly confirmed.
  • the am003 strain cultured in pdb liquid was sprayed in pots in which pepper seedlings were planted at a concentration of 10 6 to 10 7 cfu/ml more than 5 times every 3 to 5 days to show the growth state of the pepper seedlings. was observed and confirmed.
  • Figure 5f is the result of confirming whether pathogenicity of the am003 strain occurred in pepper seedlings after inoculation of the Irpex lacteus am003 strain into pepper pot seedlings, and it was confirmed that the seedlings grew more robustly and were not pathogenic.
  • Figure 5g is the result of checking the inside of the stem by cutting the epidermis of the pepper stem to determine whether pathogenicity of the am003 strain occurred in pepper seedlings after inoculation of the Irpex lacteus am003 strain into pepper pot seedlings, confirming that the seedlings grew more robustly and were not pathogenic. did.
  • the pepper seedlings showed no pathogenic phenomenon at all and showed a more active growth state due to the am003 strain.
  • the am003 strain had an excellent pathogen control effect and was not at all harmful to the host plant, pepper crops.
  • only pathogens have a mechanism that selectively acts in the same way as natural enemies.
  • the am003 strain of the present invention is 9 types (apple, pepper anthracnose, pepper blight, pepper wilt, gray mold disease, lettuce sclerotia, basil sclerotia, earthy rot sclerotia, rice leaf sheath blight) ) was confirmed to have a control effect on plant diseases.
  • the present inventor conducted the following experiment using pepper anthracnose as a surrogate probe to determine whether the strain of the present invention was effective in open fields.
  • Myeoncheon open field test field 603-2, Jagari, Myeoncheon-myeon, Dangjin-si, Chungcheongnam-do (1,200 m 2 )
  • the open-field pepper test plots above were sprayed regularly every 10 to 20 days as shown in the photos of Figures 6a to 6f, and the am003 cultured strain was sprayed on the soil about once before planting pepper seedlings. It has improved its own disease control and soil strength.
  • the laboratory-verified Irpex lacteus am003 strain was cultured in pdb liquid medium from January to March, then diluted 200 to 400 times in groundwater that passed water quality standards, and then cultured in the pepper fruits, leaves, stems, and soil of the test plot. Spraying was carried out regularly 8 to 9 times (April 26 to August 26).
  • Figure 6a is a stock solution of cultured strain of Irpex lacteus am003.
  • Figure 6b is a photograph of the blending operation for homogenization of the mycelia of the am003 stock culture strain.
  • Figure 6c shows the 200- to 400-fold dilution process of the homogenized am003 cultured strain.
  • Figure 6d is a photograph of the homogenized and diluted am003 cultured strain prepared for spraying in an open field pepper field.
  • Figure 6e is a photograph of the spraying process of am003 diluted cultured strain in an open field pepper field.
  • Figure 6f shows the production of pesticide-free clean peppers from open field pepper fields by regular (10 to 20 days) administration of the am003 strain.
  • Peppers were harvested after August 26, 2021, and as shown in Figure 6f, it was confirmed that the quality of the peppers was excellent and that anthracnose and other diseases rarely appeared. It has been repeatedly observed that by fertilizing the am003 strain from the soil before seedling during the farming season, planting seedlings, and spraying regularly, clean agricultural products can be produced as eco-friendly pure microorganisms without any chemical pesticides. In addition, the strong odor of chemical pesticides or general biological pesticides is a serious problem, but the pure microbial preparation of the am003 strain was confirmed to have a good function as an additional agricultural effect by spreading a very good scent when sprayed on farmland.

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Abstract

La présente invention concerne : une nouvelle souche de Irpex lacteus ayant les excellents effets de lutte contre l'anthracnose chez les plantes, le phytophthora brillant, la fusariose froide, la pourriture grise, la sclérotiniose de la laitue, la pourriture sclérotique du basilic, la pourriture blanche et la brûlure de la gaine du riz, et de prévention contre les virus pathogènes des plantes et les larves sur les plantes ; et des applications industrielles de celle-ci, et, plus spécifiquement, une souche de Irpex lacteus, qui est une souche de Irpex lacteus am003 ayant le numéro d'accès KACC 83059 BP, et son utilisation industrielle.
PCT/KR2023/003692 2022-03-29 2023-03-21 Souche d'irpex lacteus am003, effet de lutte contre la maladie des cultures provoquée par celle-ci, et applications agro-industrielles Ceased WO2023191365A1 (fr)

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