WO2023191365A1 - Irpex lacteus am003 strain, crop disease control effect caused thereby, and agro-industrial applications - Google Patents

Abstract

The present invention relates to: a novel Irpex lacteus strain having the excellent effects of controlling anthracnose in plants, phytophthora bright, fusarium wilt, gray mold, lettuce sclerotinia rot, sclerotinia rot on basil, white rot and rice sheath blight, and preventing against plant pathogenic viruses and larvae on plants; and industrial applications thereof, and, more specifically, to a Irpex lacteus strain, which is an Irpex lacteus am003 strain having accession number KACC 83059BP, and industrial use thereof.

Description

Ilpex Latheus AM003 strain and its crop disease control effects and agricultural industry applications

The present invention relates to Ilpex lateus strains having plant pest prevention effects and their uses.

There are still very few research results and patents at home and abroad on major diseases such as anthracnose, late blight, wilt, sclerotia, fungal disease, and leaf blight of crops.

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. There are two types of anthracnose, which causes disease in both green and red fruits, and black anthracnose, which rots mainly by forming black dots on red fruits.

In particular, 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℃, 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. In the early stage of the outbreak, water-soaked circular spots appear on the fruit and gradually expand into a circular shape. When the disease progresses severely, light yellow spore masses are formed and the fruits or stems dry out and die. (George N.Agrios, Plant pathology, World Science, 5th edition, p. 483-487, 2006)

Plant anthracnose as described above has been achieved through the treatment of numerous pesticides. However, the continued use of 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. Meanwhile, after the Rio Declaration at the UN Conference on Environment and Development in 1992, efforts were made to protect the global environment, and with the establishment of the WTO, 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. To solve these problems of using chemical pesticides, biological pesticides that use microorganisms themselves or their functions directly and indirectly have recently been attracting attention.

“Biological 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.

Accordingly, attempts have been made to use biological pesticides instead of conventional chemical pesticides for plant anthracnose as described above. In particular, microbial treatment using the genus Bacillus or Streptomycess has been mainly used to control plant anthracnose.

In order to reduce damage from pesticides and promote clean organic farming, many types of bacteria such as Bacillus have been tested, but the research was limited to fragmentary studies. Accordingly, improvement of control technology and various research and development of microorganisms in the Earth's ecosystem are necessary, and the dedicated stage of industrialization appears to be still far away. Moreover, biological pesticides for exclusive use of microorganisms have not yet been registered and are almost non-existent on the market. There are only a few patented and similar bio-pesticides, but their effectiveness is weak and not competitive with existing chemical pesticides, and the development of the right microbial balancer is still lacking. Most crops suffer from more than 10 types of major pests and diseases due to common pathogens (molds, bacteria, viruses, etc.), so if these are dealt with correctly, all pests and diseases can be prevented and controlled. 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.

Accordingly, the need for microbial-specific bio preparations, that is, keystone microbial species and microbiome, that will lead to more accurate and powerful eco-friendly organic farming is becoming important. Currently, the market is looking forward to eco-friendly bio pesticides.

[Prior art literature]

[Patent Document]

Chinese Patent CN 112899171 A

US Patent US201800200224A1

Korean Patent No. 10-2235096

[Non-patent literature]

J. Mushrooms 2021 September, 19(3):115-125 http://dx.doi.org/10.14480/JM.2021.19.3.115 Print ISSN 1738-0294, Online ISSN 2288-8853

Against the above background, 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. In one aspect of the present invention, 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 groundbreaking keystone species strain from the natural world to prevent the occurrence of plant diseases, including plant anthracnose. We aim to provide methods to fundamentally cure and prevent.

In addition, 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.

In addition, 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.

There are no related patents that can control the above 9 types of diseases simultaneously. In addition, one aspect of the present invention seeks to provide a groundbreaking 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.

The object of the present invention is not limited to the objects mentioned above. The object of the present invention will become clearer from the following description and may be realized by means and combinations thereof as set forth in the claims.

In order to achieve the above objective, the following solution is provided.

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.

According to one aspect of the present invention, 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.

In one aspect of the present invention, 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.

In one aspect of the present invention, the plant anthrax is provided as a strain selected from the genus Glomerella and Colletotrichum genus Anthrax, and 9 types of disease pathogens.

In one aspect of the present invention, the strain includes the DNA barcode marker base sequence of ITS 1 and 2 of the rDNA structure (nuclear ribosomal RNA cistron).

In one aspect of the present invention, the strain is Irpex lacteus am003 ( Irpex lacteus am003).

In one aspect of the present invention, the strain provides a strain whose accession number is Irpex lacteus am003 (KACC 83059BP).

In one aspect of the present invention, 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; Provided is 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.

In one embodiment, 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 to the Irpex lacteus am003 strain that can control gray mold disease and rice leaf sheath blight, and provides control methods for 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; In addition to 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.

According to one aspect of the present invention, 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. On the other hand, little research has been done on crop disease control yet, but in the present invention, 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 groundbreaking 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. As a result, 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.

The effects of the present invention are not limited to the effects mentioned above. The effects of the present invention should be understood to include all effects that can be inferred from the following description.

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). Specifically, 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. Specifically, 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 above objects, other objects, features and advantages of the present invention will be easily understood through the following preferred embodiments related to the attached drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed content will be thorough and complete and so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art.

In this specification, terms such as “comprise” or “have” are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the specification, but are not intended to indicate the presence of one or more other features. It should be understood that this does not exclude in advance the possibility of the existence or addition of elements, numbers, steps, operations, components, parts, or combinations thereof.

In this specification, 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. For example, 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. Also, for example, 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.

Hereinafter, the present invention will be described in detail.

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. In addition, 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.

Red pepper blight outbreak period and symptoms

It is the most damaging disease in peppers and is considered one of the two major diseases of peppers along with anthracnose. It occurs from mid-to-late May immediately after field planting and becomes rapidly prevalent after the rainy season in July. The disease is caused by invasion of the Phytophthora capsici (Leonian) mold, which belongs to the oomycete family. There are 12 known types of blight fungi, but including those that are likely to occur in the future, there are about 20 types. Plague bacteria form thread-like hyphae without septa and spores of various shapes. Among them, the most important are conidia, which are round or oval in shape and contain zoospores with two swimming hairs. These zoospores swim along the water to reach the host and invade the plant. When the surrounding environment deteriorates in the late stages of crop growth, 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.

In early July, during the first flowering period of peppers, the phloem of the rhizomes exposed at 1 to 3 cm above the surface is infected with germs. In the early stages of the disease, dark green, water-soaked lesions appear. The lesion area is slightly depressed. In the later stages of the disease, it gradually turns black brown, the stem and leaves dry out green, and the pepper becomes wilted. It takes 5 to 6 days for the disease to develop and progress. After a week, the diseased trees dry completely and the peppers cannot be harvested.

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 )

It belongs to the Ascomycetes and mainly forms conidia, but in rare cases it also forms ascospores.

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℃, 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.

Pepper - wilt disease

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.

Rice sheath blight pathogen Thanatephorus cucumeris

When the sclerotia that have survived the winter in the soil, rice straw, or stumps are irrigated, tilled, and harrowed before planting, they float on the water, and after planting, they attach themselves between the leaves and leaf sheaths.

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

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.

Lettuce sclerotinia rot pathogen Sclerotinia sclerotiorum

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.

Disease occurrence is severe in high humidity and cool temperatures of 15 to 25°C.

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.

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. Disease occurrence is severe in high humidity and cool temperatures of 15 to 25 degrees Celsius. (Sclerotinia caused by Sclerotinia sclerotiorum and Sclerotinia minor in lettuce grown in facilities)

basil sclerotia

For basil sclerotia disease, the pathogen was isolated from lesions on diseased leaves and stems collected from the field, and mycological characteristics and pathogenicity were tested. Additionally, the pathogen was identified by analyzing the internal transcribed spacer (ITS) sequence of rDNA. Diseases occurring in basil around the world have been reported to include gray mold ( Botrytis cinerea ), anthracnose ( Colletotrichum gloeosporioides ), wilt ( Fusarium oxysporum ), stem rot ( Rhizoctonia solani ), sclerotia ( Sclerotinia sclerotiorum ), and pythium ultimum ( Pythium ultimum ). (Garibaldi et al., 1997).

Basil ( 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

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. Under these characteristics and conditions, 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. Provides microbial preparations that

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. , 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.

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 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.

Hereinafter, various aspects of the present invention will be described.

One aspect of the present invention provides an Irpex lacteus strain that has antibacterial activity against plant pathogenic microorganisms and plant pathogenic viruses.

According to one aspect of the present invention, 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.

In one aspect of the present invention, 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.

In one aspect of the present invention, 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. .

In one aspect of the present invention, the plant pathogenic virus is a tomato spotted wilt virus (TSWV) strain.

In one aspect of the present invention, 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 , and the bacteria causing plant sclerotia are The bacterium is a genus of Scerotinia , the plant fungal disease-causing bacterium is a genus of Botrytis , and the bacterium causing leaf sheath blight is a genus of Thanatephorus .

In one aspect of the present invention, 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.

In one aspect of the present invention, the strain is Ilpex Latheus am003.

In one aspect of the present invention, 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.

In the second aspect of the present invention, 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; Provided is 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.

In the third aspect of the present invention, 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.

In the fourth aspect of the present invention, 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; Provided is 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.

In the fifth aspect of the present invention, 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.

In the sixth aspect of the present invention, 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; Provided is 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.

In the seventh aspect of the present invention, 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; Provided is a composition for controlling leaf sheath blight, comprising one or more of the following.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1. Isolation of crop pest control microorganism am003 strain

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.

Taking advantage of these characteristics, first, 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.

The isolation area was purely isolated from white rot fungi from a mountainous forest area in Gyeonggi-do. Figure 1 is a photograph of an isolated strain after culturing it in PDA medium for 1 to 2 weeks.

Example 2. Morphophysiological identification of am003 strain

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)

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.

After pure passage of the white fungus isolated from the mountainous forest area of Gyeonggi-do as in Example 1 above, 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).

ITS region gene sequence analysis and homology comparison

PCR amplification of ITS region gene base sequence by colony PCR method

Primer used for ITS region amplification

ITS1(5 <sup>`</sup> -TCCGTAGGTGAACCTGGCGG-3` )

ITS4(5`-TCCTCCGCTTATTGATATGC-3` )

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).

ITS region gene sequence homology search

〔NCBI BLAST Search: http://blast.ncbi.nlm.nih.gov/Blast.cgi〕

To create a phylogenetic tree, 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.

18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1 and 5.8S ribosomal RNA gene, complete sequence; and internal transcribed spacer 2, partial sequence

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.

For 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.

In more detail, ITS rDNA (DNA sequence that code for ribosomal RNA) is extracted, the related PCR nucleotide (Universal primer) is used to amplify the gene, and when this is electrophoresed and read with a decoder, the DNA sequence appears and displays the sequence. This is the sequence below.

Then, when this sequence is grafted onto the NCBI data base, the genus and species can be estimated by comparing homology with existing strains, and it is already a standardized global standard identification and classification method. The ITS 1 sequence of the am003 strain analyzed according to the above experiment was the same as the sequence in Sequence Listing 1 below.

[Example 1 Identification and classification of strains]

Sequence Listing 1: ITS region sequence Irpex lacteus am003 ITS sequence

Irpex lacteus am003strain 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1

GGCCTCTCACGAGGCATGTGCACGCCTGGCTCATCCACTCTTAACCTCTGTGCACTTTATGTAAGAGAAAAAAATGGTGGAAGCTTCCAGGATCTCGCGAGAGGTCTTCGGTTGAACAAGCCGTTTTTCTTTCTTATGTTTTACTACAAACGCTTCAGTTATAGAATGTCAACTGTGTATAACACATTTATATACAACTTTCAGCAACGGATCTCTTGGCTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAG TAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACCTTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTGAGTCTCATGGTATTCTCAACCCCTAAATTTTTGTAATGAAGGTTTAGCGGGCTTGGACTTGGAGGTTGTGTCGGCCCTTGTCGGTCGACTCCTCTGAAATGCATTAGCGTGAATCTTACGGATCGCCTTCAGTGTGATAATTATCTGCGCTGTGGTGTTGAAGTATTTATG GTGTTCATGCTTCGAACCGTCTCCTTGCCGAGACAATCATTTGACAATCTGAGCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATCAAT

Re-verification process of Irpex lacteus standard strain FD-9 and am003 strain

After analyzing the sequence of strain 003 above, it showed a 99.3% affinity relationship with the standard strain Irpex lacteus FD-9 (sequence list 2) and molecular phylogenetic analysis, and was identified as a strain of the Irpex lacteus strain (Figure 4c schematic).

Sequence Listing 2: ITS region sequence Irpex lacteus FD-9 ITS sequence

Irpex lacteus strain FD-9 18S ribosomal RNA gene, partial sequence; internal transcribed spacer 1

GenBank: KP135026

CAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTATCGAGTTTTGAACGGGTTGTAGCTGGCCTCTCA

CGAGGCATGTGCACGCCTGGCTCATCCACTCTTAACCTCTGTGCACTTTATGTAAGAGAAAAAAAATGGTG

GAAGCTTCCAGGATCTCGCGAGAGGTCTTCGGTTGAACAAGCCGTTTTTCTTTCTTATGTTTTACTACAA

ACGCTTCAGTTATAGAATGTCAACTGTGTATAACACATTTATATACAACTTTCAGCAACGGATCTCTTGG

CTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCG

AATCTTTGAACGCACCTTGCACTCCTTGGTATTCCGAGGAGTATGCCTGTTTGAGTCTCATGGTATTCTC

RACCCCTAAATTTTTGTAATGAAGGTTTAGCGGGCTTGGACTTGGAGGTTGTGTCGGCCCTCGCYGGTCG

ACTCCTCTGAAATGCATTAGCGTGAATCTTACGGATCGCCTTCAGTGTGATAATTATCTGCCGCTGTGGTG

TTGAAGTATTTATGGTGTTCATGCTTCGAACCGTCTCCTTGCCGAGACAATCATTTGACAATCTGAGCTC

AAATCAGG

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.

As a result of the analysis, the strain in Sequence Listing 1 had 100% identical ITS 1 region sequence, as shown in Figures 4a and 4b, confirming the pure identity of the strain.

(The partial sequence of internal transcribed spacer 1; the complete sequence of the 5.8S ribosomal RNA gene and internal transcribed spacer 2; and the partial sequence of the large subunit ribosomal RNA gene were repeatedly confirmed to be identical.)

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.

Meanwhile, the strain was deposited with the National Institute of Agricultural Sciences on February 9, 2022, and the accession number was determined to be KACC 83059 BP.

Example 4. Confirmation of the inhibitory effect of am003 strain on 9 types of plant pathogens on PDA (potato dextrose agar) medium

To determine whether 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), an experiment was conducted as follows. proceeded.

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.

The experimental results were the same as Figures 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.

The mutual reaction between the standard strain of the pathogen and the am003 strain, as shown by the proliferation of white mycelia of am003, 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.

Example 5. Test to confirm pathogenicity of am003 strain on pepper plants in pepper pot seedlings

As shown in Figures 5f and 5g, 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.

The experimental results were the same as Figures 5f and 5g.

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.

As shown in Figures 5f and 5g, the pepper seedlings showed no pathogenic phenomenon at all and showed a more active growth state due to the am003 strain. As a result, it was confirmed that the am003 strain had an excellent pathogen control effect and was not at all harmful to the host plant, pepper crops. In other words, it was confirmed that only pathogens have a mechanism that selectively acts in the same way as natural enemies.

Example 6. Field test of am003 strain

As confirmed in Examples 4 to 5, in the laboratory unit, 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.

Accordingly, 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 )

Local weather conditions: Data from the Korea Meteorological Administration’s Meteorological Data Open Portal (Daejeon Regional Meteorological Administration)

date
2021.4.26~8.26 Average temperature (℃) Daily precipitation (mm) Average wind speed (m/s) April 26, 2021 14.4 0 1.4 May 13, 2021 19.5 0 1.2 June 7, 2021 20.4 0 One June 22, 2021 22 0 One July 6, 2021 23.7 9 0.7 July 13, 2021 26.6 0 0.8 July 23, 2021 27.8 0 0.8 August 3, 2021 26.8 0 0.8 August 16, 2021 25.5 0 0.9

2021. Starting from mid-April, 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. Under these weather conditions, 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).

Photos of the scene were the same as Figures 6A to 6F.

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.

[Accession number]

Name of depository institution: Korea Agricultural and Biotechnology Research Institute

Accession number: KACC83059BP

Trust date: 20220209

Claims (27)

Irpex lacteus strain with antibacterial activity against plant pathogenic microorganisms and plant pathogenic viruses. According to paragraph 1, The strain is a highly heat-resistant strain that can survive at a temperature of 110°C or higher and for a time of more than 30 minutes. According to paragraph 1, The plant pathogenic microorganism is a strain that is at least one of plant anthrax, plant blight, plant wilt-causing bacteria, plant sclerotia-causing bacteria, plant fungal disease-causing bacteria, and leaf spot blight-causing bacteria. According to paragraph 1, The plant pathogenic virus is a tomato spotted wilt virus (TSWV) strain. According to paragraph 3, The plant anthrax is anthrax of the genus Glomerella and One or more selected from the anthrax bacteria of the genus Colletotrichum , The plant blight bacteria are of the genus Phytophotora , The plant wilt disease-causing bacteria are Fusarium genus, The plant sclerotia-causing bacteria are bacteria of the genus Scerotinia , The plant fungal disease-causing bacteria are Botrytis genus, The bacteria causing leaf sheath blight are members of the genus Thanatephorus , strain. According to paragraph 1, The strain includes the DNA barcode marker base sequence of ITS 1 of 18S of the rDNA structure (nuclear ribosomal RNA cistron) of Sequence Listing 1. According to paragraph 1, The strain is Ilpex Latheus am003. According to paragraph 1, The strain has an accession number of KACC 83059BP. The strain according to any one of claims 1 to 8; 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 containing one or more of the following: The strain according to any one of claims 1 to 8; 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 method for controlling plant anthracnose comprising: using one or more of the following. According to clause 10, The above plants include pepper, persimmon, apple, pear, grape, ginseng, deodeok, bellflower root, rice, barley, wheat, corn, Schisandra chinensis, wolfberry, blueberry, persimmon, 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, plum, asteraceae, orchid , a method for controlling plant anthracnose, which is one or more of flowering plants, coffee trees, pine trees, and pine trees. The strain according to any one of claims 1 to 8; 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 strain according to any one of claims 1 to 8; 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 method for controlling plant blight comprising: using one or more of the following. According to clause 13, The plant blight is caused by bacteria of the genus Phytophotora , The plant blight is pepper blight, a method for controlling plant blight. The strain according to any one of claims 1 to 8; 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 blight, comprising one or more of the following. The strain according to any one of claims 1 to 8; 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 method for controlling plant wilt disease comprising: using one or more of the following. According to clause 16, The plant wilt disease is caused by fungi of the genus Fusarium , A method for controlling plant wilt disease, wherein the plant wilt disease is pepper wilt disease. The strain according to any one of claims 1 to 8; 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 strain according to any one of claims 1 to 8; 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 method for controlling plant sclerotia disease comprising: using one or more of the following. According to clause 19, The plant sclerotia disease is caused by bacteria of the genus Scerotinia , The plant sclerotia disease is any one or more of soil rot sclerotia, lettuce sclerotia, and basil sclerotia, a method for controlling plant sclerotia. The strain according to any one of claims 1 to 8; 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 sclerotia, comprising one or more of the following. The strain according to any one of claims 1 to 8; 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 method for controlling plant fungal diseases comprising: using one or more of the following. According to clause 22, The plant fungal disease is caused by bacteria of the genus Botrytis , A method for controlling a plant fungal disease, wherein the plant fungal disease is gray mold disease. The strain according to any one of claims 1 to 8; 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 strain according to any one of claims 1 to 8; 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 method of controlling leaf sheath blight comprising using one or more of the following. According to clause 25, The leaf sheath blight is caused by bacteria of the genus Thanatephorus , The leaf sheath blight is a method of controlling leaf sheath blight, which is rice sheath blight. The strain according to any one of claims 1 to 8; 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.

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