KR100319135B1 - Microbiological preparation inhibiting plant diseases - Google Patents

Abstract

The present invention relates to a microbial agent having a antagonistic effect on plant diseases, and to a microbial agent having an antagonism on a plant disease formulated therein, and to a method for preparing the same. Genie is a microbial agent having plant disease antagonism prepared by growing and developing Bacillus subtilis CP220 (Accession No. KCTC-8831P) in a medium and diluting the dilution at about 100 to 1,000 times. will be.

Description

Microbiological preparation inhibiting plant diseases

The present invention relates to a microbial agent having a antagonistic effect on plant diseases, and a microbial agent having an antagonism on plant diseases formulated therewith and a method for producing the same.

There are 4,157 pests that cause damage to crops grown in Korea, but 1,199 species (bottle 574 and 625 pests) have been confirmed to occur. It is bringing reality.

As these pests cause damage to crops, one of the easiest means to exclude or control them from crops is called pesticides, which are recognized as natural when pests and weeds occur on crops. It is. However, as pesticides, which are convenient control measures, have recently been raised, such as safety issues, residual problems (crops, soil, environment), various problems on the natural ecosystem, and pollution, the negative view tends to increase gradually.

In view of the fact that even with these various problems, it is impossible to prevent the use of all pesticides currently in use, studies on biological control methods using low-toxic, safe synthetic pesticides or microorganisms, which are safer and have fewer side effects than current pesticides, have been conducted in each country. Has been.

The research history of controlling pests using microorganisms was the first attempt to control beetle pests with a fungus in Germany in 1880. Since then, many studies have been carried out and about 70 species have been put to practical use as microbial pesticides worldwide.

Looking at the current state of development in foreign countries, many researches have been attempted as biopesticides using microorganisms themselves. This is an attempt to control pests using microorganisms itself. Representative studies using antagonistic microorganisms are representative.

In 1927, actinomycetes were first used in the United States to control potato beetle disease, and in 1951, a study was carried out on the control of viruses using cross-protection and sweet potato stem rot using fungi.

On the other hand, in the study using various natural microorganisms killing tissues, organs and whole or paralyzed by toxins contained in microorganisms, various microorganisms have been used for controlling since 1921, which are shown in Table 1.

History of research on pest control using microorganisms division Kinds Start year Developing country Prevention target Antagonist Three Fungal Fungal Viruses 1968193219271955 United States of America United States of America Corn seedling disease pea seedling disease potato Cross defense Three fungal viruses 196419511951 United States of America Potato Blue Bottle Sweet Potato Stem Rot Disease CTV Natural enemies Three Fungal Fungal Viruses 1921188019561928 Germany Germany Daily Lepidoptera pest coleoptera pest pest

It is known that 16 kinds of pest control and 50 kinds of pest control are known to be practically used as biological pesticides to the world by using microorganisms for controlling pests, and many microorganisms are being promoted for practical use.

Bacteria control agents using microorganisms have been put to practical use in the form of pesticides since 1960.In Japan, many products have been used since 1962 as a probiotic for tobacco waist blight Trichoderm a. It is noted that it was developed for use. These commercialized products are shown in Table 2.

Microbial fungicides for pest control microbe Yong Yong Lee Target disease product name Registrar Year Germ Agrobacterium radiobacter strain 84 A. radiobacter K1026 Bacillus subtilis Pseudomonas cepacia Pseudomonas fluorescens EG-1053 Streptomyces griseovirides Crown Gothic BoneSeedling Root Disease Infection seed-bornSeedling rootDamping-off Mystery Disease ( Fusarium Alternaria etc.) GalltrolBakuterozuDygallNogallQuantum 4000GUS 2000Blue circleDagger GMycostop United States ('79) Japan ('89) CanadaAustraliaUnited StatesUnited StatesUnited StatesUnited States ('88) United States mold Gliocladium virens GL-21 Pythium ligandam T. harzianum Rifaistrain KRL-AG 2 T. harzianum / poly- sporum T. lignorum Trichoderma viridae Temple jalrokbyeong (Rhizoctonia, Pythium) Sugar beet disease . Jalrokbyeong Temple (Pythium) Wood-decaying fungi Southem blightSore shin (Tobacco) Verticillium in Matsutake Plum silver leaf disease WRC-GL-21-WRC-AP-1PolygandronF-StopBinab TTrichoderma (spore) BINAB TSEPPIC BINAB United States ('90) Czech SlovakiaUnited StatesUnited StatesJapan ('62) FranceUnited Kingdom

The most innovative and successful products for controlling pesticides are Agrobacterium radioacter strain 84 and K1026, which are antagonistic microorganisms to the root gall that occurs in the roots of various crops, especially perennial woody roots. Galltrol, Dygall, Nogall, Bakuterozu products using the strains.

The organic synthetic pesticides currently used have a low control effect on all bacterial diseases, but especially the root-knot bacterial diseases cannot be effective, whereas the organic pesticides have a definite effect and are used as excellent control agents.

The control mechanism of this biopesticide has been shown to inhibit the cell wall synthesis of pathogens by an antimicrobial agent called Agrocin, produced by the A. radiobacter bacterium.

On the other hand, the microorganisms used in the pest control field are molds, bacteria, viruses, and many products are mite, aphids, root-knot nematodes, scarabs, Chinese cabbage moths, green chestnut moths, pine needles, leaf bugs, and greenhouse powder. It is put to practical use. As the most well-known of these products is Bacillus Tudor Rigi language Enschede in the Bt products using Bt bacteria (Bacillus thuringiensi s less Bt), starting with the 1921 study history's first microbial pesticides worldwide since the product 1948 practical It is a famous product that occupies about 90 or more of microbial pesticides .

Bt bacteria are many variants, and each variant of the production is known to be different from the control target pests by a toxin, a subspecies of Bt Kur Starkey (kurstaki), Aizawa (aizawai), Israel sheath (israensis), San Diego (san diego), tenebrionis ( tenebrionis ), etc. are used, and the types of products are mixed with bacteria and toxins and toxins themselves.

The insecticidal period is known to cause toxins to be activated by strong alkaline digestion of carcasses and destroy the cells of the intestine. Recently, many researches on the use of genetic engineering techniques and breeding of insecticidal crops have been actively conducted.

Among the microbial pesticides introduced into Korea and being used in practice , products using Bt subspecies kurstaki and aizawai strains were registered in 1981, and have been used as pests for white fire moths since 1989. The outbreak of Chinese cabbage moth and drug resistance of organic synthetic pesticides is known as a fast-growing product with shipments of 120 tons (sales of 3.8 billion won) in 1995. Recently, Bt agrochemicals are also produced and supplied in Korea.

When the microorganism is artificially cultured, the metabolites produced by the microorganism are discharged into the culture medium. The metabolic active substances are separated and purified and used as antibiotics for medicinal and agricultural use, and more than 8,000 kinds of these antibiotics are known. There are about 600 species, and about 20 species are used for agriculture, 6 species are widely used for pest control, and 3 species are used for pest control.

Agricultural antibiotic-producing bacteria are known as Streptomyce s strains among Actinomycetes , which are widely distributed in soil. In 1958, the world's first agricultural antibiotic was led by Bratisicidin S, known as an antibiotic for control of bleeding diseases, with Kasugamycin, Polyoxin, and Validamycin. Tetraactin, Avermectin, and Milbemectin, which have been used for control and are used as insecticides for mite control, have been developed.

On the other hand, if you look at the development status in Korea, it has been studied for the prevention of diseases using microorganisms from 1985 pathologists of national research institutes and universities, TMV, Bacterialwilt of cucumber, Fusarium wilt of cucumber, Phytophthora blight of pepper , Fusarium wilt of strawberry, Rhizoctonia bud rot, Damping-off of sugar beet, rice blasting, control of paperback disease, etc., are reported in Table 3.

Control of Crop Diseases Caused by Microorganisms in Korea crops Target Bottle Microorganism types Report year tobacco TMVBacterial wilt Virulence virusNon pathogenic P. solanacearum '85'85 cucumber Fusarium wilt Rhizoctonia antagonists Pseudomonas gladioli Non pathogenic strain of Fusarium oxysporum f. sp. cucumerinum Gliocladium virens Pseudomonas putida '87'92'93'95 pepper Phythophthora blight Bacillus sp. (AC-1) Pseudomonas cepacia Tricoderma harzianum Enterobacter agglomerans Non paghogenic strain of Phytophthora capsici '86'88'89'92 Strawberry Fusarium wiltRhizoctonia bud rot Tricoderma sp. T. harzianum Pseudomonas gladioli Antagonistic microorganisms '88'95'90'94 sugar cane Mystery Disease Pseudomonas sp. '88 rice plant Blast, sheath blight Pseudomonas sp. '90

The use of microorganisms in pest control has been reported to isolate the fungus for controlling root nodules and pine needles, and cabbage butterfly using nuclear polyhedron virus, and tobacco moth using cellular polyhedron virus.

Research on pest control using microorganisms in Korea object Microorganisms used Report year Root-kont nematode Fungi (isolated from infected indect) '88 Tobancco cutworom Nuclear polyhedrosis Virus '89 Fall webworm Nuclear polyhedrosis Virus '89 Common cabbage worm Granulosis Virus '91 Armyworm Nuclear polyhedrosis Virus '91 Oriental tobacco budworm Cytoplasmic polyhedrosis Virus '91 Pine leaf gall midge Stterive fungus '95

In particular, the reported results are utilized possibly higher quality known as Bacillus (Bacillus) for pepper blight control in strain strain AC-1 during and pest control Bacillus Tudor Rigi N-Sys for (Bacillus thuringiensis) and Entebbe and Beria Bridge Ana ( Beauveria bassiana ) strains have been patented and partly used, and non-pathogenic strains for controlling cucumber vine cutting disease and powdery mildew parasites in various crops are patented and considered to be practical.

Meanwhile, research on agricultural antibiotics, which is an active substance for producing microorganisms, was also carried out 20-30 years later than in foreign countries, and is still searching for new materials. As a result, we have identified and identified cycloheximide for controlling rice leaf blight, and Maculocin by Streptomyces. However, new material isolation or promising The utilization of the substance is not reached.

According to the '92 -93 survey by the Institute of Agricultural Technology, 167 types of pathogens were investigated in 18 orchards, including apple trees and pears, in fruit orchards and neglected cultivation management. 11 species, 5 viruses, and 3 others. Apples were found to be ovules, layered rots, brown patterned, spotted deciduous, and red star patterned diseases, and black star pattern, pseudo black pattern disease, and black pattern disease. The occurrence of red star pattern disease was serious.

The types and incidences of fruit trees appear to be different depending on the season of growth, variety, and region.Seasonal disease, gray mold, spear disease, red star pattern disease, bacterial pore disease, peach organ disease, plum porcelain disease in spring There were a lot of bottles that like relatively low temperature and humidity conditions. Most bottles, however, like hot and humid environments, so they usually start in the rainy season from August to September. In addition, diseases that occur in semi-dry conditions, such as powdery mildew and rust, are often damaged in spring and autumn.

In the recent agricultural production, the over-injection of chemical pesticides and chemical fertilizers causes severe pollution of groundwater and agricultural environment and excessive production costs, thus reducing the overuse of chemical pesticides and chemical fertilizers, improving the agricultural environment, and reducing the costs of using various agricultural by-products. There is a strong demand for the development of environmentally friendly farming methods that produce low pollution and high quality agricultural products. Such environmentally sustainable agricultural development is necessary not only for capital and technology-intensive plant horticulture, but also for the production of field and plant fruit trees.

Low-pollution and high-quality fruit production is being developed in the agricultural production method called organic method in terms of maximizing utilization efficiency of organic fertilizers using persimmon pesticides and agricultural by-products.In Korea, fermented organic compost and inorganic / organic mixed nutrient solution There is a growing interest in environmental farming, which produces high-quality and low-pollution fruits by treating the ground and underground.

However, in order for the environmental conservation farming method to be fully settled in Korea, researches on the production technology using fermented compost, the development of biopesticides that control pests biologically using sterilizing and insecticidal effective microorganisms, etc. should be conducted in-depth.

Therefore, the present invention isolates major pathogens that are currently a problem in agricultural production, and has developed antagonistic microorganisms as biological control agents.

Figure 1 shows the change in the kinase activity according to the growth curve of the antagonist B. subtilis CP220 of the present invention.

■: Kitanase activity, ●: Growth degree

Figure 2 is a photograph showing the growth inhibitory effect after spraying antagonist B. subtilis CP220 of the present invention to phytopathogens.

A: decay pathogen, B: anthrax pathogen, C: Bran pathogen, D: blue fungal pathogen,

E: Root rot pathogen, F: Gray fungal pathogen

Figure 3 is a photograph showing the growth inhibitory effect of phytopathogenic bacteria in the antagonist B. subtilis CP220 culture medium after the filtration (heat treatment and no treatment) medium of the present invention.

A: decay pathogen, B: anthrax pathogen, C: Bran pathogen, D: blue fungal pathogen,

E: Root rot pathogen, F: Gray fungal pathogen

Figure 4 is a diagram showing the survival rate after spraying the antagonist B. subtilis CP220 KS of the present invention to apple leaves.

5 is an illustration of the survival rate of the antagonist B. subtilis CP220 KS of the present invention after spraying anthracol, Bordeaux pesticides on apple leaves.

●: Anthracol, ▲: Bordeaux

Figure 6 is a photograph showing the inhibitory effect of rot antagonist B. subtilis CP220 of the present invention in the apple fruit rot, anthrax, blue fungal disease.

A: No antagonist treatment, B: Pathogen treatment after antagonist addition

Figure 7 is a photograph showing the antagonistic B. subtilis CP220 of the present invention inhibiting pot cultivated apple ovule disease.

VC: pathogen only, A: antagonist after pathogen treatment,

B: Antagonist application after pathogen treatment

8 is a photograph showing the antagonistic B. subtilis CP220 of the present invention inhibits potted apple anthrax.

A: antagonist free after pathogen treatment, B: antagonist spray after pathogen treatment,

C: Pathogen treatment after antagonist application

Therefore, an object of the present invention is to grow and develop Bacillus subtilis CP220 (Accession No. KCTC-8831P) in a medium having an antagonism against plant diseases isolated from nature, dilution ratio of about 100 to 1,000 It is to provide a microbial agent having a plant disease antagonist prepared by diluting about twice.

At this time, the plant pathogens that can be applied to the microbial preparations include rotted rot (rot), anthrax, spotted deciduous disease (spot deciduous disease), ovule disease, stem blight (dongdongjeung), sesame seedlings, wilted disease, swelling disease (blotting disease), It is characterized by one or more plant diseases selected from pathogenic fungi of gray mold disease, root rot disease, purple-wing disease, and white-wing disease.

In addition, the isolates of this strain are plant rot (rot), anthrax, spotted deciduous (spot deciduous), ovule, stem blight (dongdongjeung), sesame, erosion, wilted disease, diarrhea (dysseal), ash fungus Antagonist against pathogenic bacteria selected from pathogenic fungi of root rot disease, purple wing disease, and white wing disease, isolated from microbial strains in nature This excellent antagonist, CP220, was isolated.

As a result, Bacillus subtilis was identified as Bacillus subtilis CP220 or Bacillus subtilis CP220, which was deposited on September 29, 1997 at KCTC-8831P in the Genetic Resource Center of Korea Institute of Science and Technology.

The optimum temperature for the growth of antagonist Bacillus subtilis CP220 was 32 ℃ and pH was around 7.0. In addition, the composition of the medium for mass production has the advantage that it is easily available as a cheap raw material in general farms, and the growth of cells is relatively strong, and soy sauce 2.5-3.5 is selected as nitrogen source and 4.5-5.5 sugar as carbon source.

As a result of measuring the enzyme activity secreted by antagonists and pathogens, chitinase secretion was observed in antagonists and pectinase activity was detected in pathogens. All of the changes in enzyme activity according to growth curve in antagonistic optimal medium condition showed growth-associated pattern in which the enzyme activity increased similarly to the growth curve pattern.

Dilution ratio was about 100 to 1,000 times for the field application of antagonistic bacteria.

The antagonists secreted by antagonists have been found to be very resistant to heat because they have antagonistic effects on rot pathogens, anthrax pathogens, ovulation pathogens, blue mold pathogens, root rot pathogens and gray mold pathogens when antagonists are sprayed and heat treated and untreated. .

Experimental results on the storage stability of isolated antagonists showed that the shelf life was the most effective when glycerol was added and the antagonistic bacteria stored for 1 year also maintained excellent antagonism. appear.

About 10 days after application of Bacillus subtilis CP220 to apple trees, about 33 microorganisms survived on the leaves. As a result, the microbes appear to survive to some extent on the leaves.

The titration time of antagonist application was investigated after spraying pesticides. When the antagonist microorganism Bacillus subtilis CP220 was sprayed 16 days after anthracol hydration, 6-12 Bordeaux solution was able to grow after 18 days of spraying. In view of this result, it is thought that antagonist spray is effective in the case of anthracol hydration after 16 days of spraying and 18 days after spraying of 6-12 Bordeaux solution.

When inoculating apple fruit into spore suspension of rot, anthrax, and blue fungus in an in vivo bioassay, 100 days of inoculation resulted in 6 days of inoculation and the diameter of the lesion was 18mm or more. On day 6, the antifungal activity was over 50.

As a result of examining the control effect of ovulation and anthrax using isolated antagonistic bacteria in pot cultivated apples, it was possible to prevent it. On the other hand, the control effect of anthrax, unlike in case of ovulation, is effective only when the antagonists are treated first.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example 1 Separation of Effective Microorganisms from Natural Systems

In order to separate effective antagonistic microorganisms with antagonistic resistance against phytopathogens, samples collected from all over the country were processed as in the experimental method, and 10,000 strains were isolated. The isolated microorganisms were inoculated in nutrient agar medium to form a flora and then refrigerated and used for antagonistic experiments with pathogens.

Example 2 Isolation and Collection of Major Plant Pathogenic Fungi

The main diseases of the plant are leaf rot (rot), anthrax, spotted deciduous (spot deciduous), ovule, stem blight (warring), sesame, rot, wilting disease, burrowing disease, gray mold, root rot, In order to isolate pathogens from infected tree leaves such as purple wing disease and white wing disease, white spots of Galgong-ri, Sinbuk-myeon, Yeongam-gun, Jeonnam Province, were investigated. Was collected to isolate the pathogen. It was also sold by the National Agricultural Science and Technology Institute (NASTI). To isolate the pathogen, the diseased surface was sterilized with 70 ethanol and 5 sodium hypochlorite (NaOCl), and the lesion was placed on a pathogen-selective medium (Potato Dextrose Agar; PDA + streptomycine 200μl / ml, pH 3.0). Place and incubate for 2 to 3 days in a 25 ℃ incubator to form a colony (colony) as shown in Table 5.

Phytopathogenic bacteria used in the experiment Plant pathogen Korean English name Host plants Botryosphaeria dothidea Var. (BD) Layered rot germ canker, die-back Apple, chestnut Glomerella cingulata (GC) Anthrax bitter rot, anathracnose Chili, apple, pear, garland chrysanthemum, persimmon, grape, fig Alternaria mali (AM) Spotted deciduous bacteria (spotted deciduous bacteria) alternaria leaf spot Apple, grape Rosellinia necatrix (RN) Whitewing Streptococcus root rot Apple, Cherry Valsa ceratosperma (VC) Inoculation canker Apple Botrytis cinerea (bc) Gray mold gray mold rot Chili, tomato, strawberry, cucumber, sweet potato, perilla leek eggplant lettuce, celery, onion bean Phomopsis sp. (PV) Stem blight bacteria die-back Plum, Jujube, Ginkgo, Apple Fusarium oxysporium (FO) Wilted germs fusarium wilt Chili, tomato, sweet potato, spinach, watermelon, loofah, cucumber, daikon, cabbage, onion, yeon, strawberry, peanut, beans, melon Pythium ultium (PU) Shrop disease damping off Pepper, Chinese Cabbage Fusarium solani (FS) Root rot surface rot Sweet Potato, Kidney Bean, Grass Helicobasidium mompa (HM) Common Wings Disease violet root rot Sweet potato

Example 3 Antagonist Microbial Screening for Various Plant Pathogens

(1) Selection of antagonists against pathogens using isolated microorganisms

Table 6 shows the results of antagonistic experiments on phytopathogenic bacteria using isolated antagonistic microorganisms for biological control of apple pathogenic fungi. As a result of the antagonistic test on the phytopathogenic bacteria isolated from this study, the superior antagonism of the antagonist microorganism CP220 was in the order of the disease of wormwood (loose disease)> wilting disease> winged disease (white door disease)> ovule disease> root rot disease> gray mold disease> stripe pattern The antagonists were excellent in the order of rot (rot), spotted deciduous disease (spot deciduous disease), anthrax, frequent wing pattern disease (adult disease)> bundling disease> blue mold disease.

Inhibitory Effect of Antagonist CP220 on Phytopathogenic Bacteria Pathogen P.ultium F.oxysporium ( withering disease) R. necatrx ( white door disease) V. ceratosperma (ovulation) F. solani (root rot) B. cinerea (ash fungus) CP220 93 82 64 60 60 60

Pathogen B. dothidea ( corruption ) A. mali (spot deciduous) G. cingulata (anthrax) H. mompa Phomopsis sp . P.expansum ( blue fungal disease) CP220 57 53 50 50 21 11

NT; Colony diameter of no treatment (mm)

T; Size of colony [colony diameter of treatment (mm)]

Example 4 Identification of Antagonists

In order to identify CP220, an antagonistic strain against phytopathogenic bacteria, morphological characteristics, culture characteristics and physiological biochemical properties of microorganisms were examined. Based on these results, according to the classification criteria described in Bergey's manual of systematic bacteriology and microbiological methods, the CP220 strain was estimated to be Bacillus subtilis or its flexible bacteria. This antagonist microorganism has been deposited with the Institute of Biotechnology (Accession No .: KCTC 8831P).

Example 5 Mass Growth of Antagonistic Microorganisms

In order to apply the selected antagonists to the actual field experiments, high concentration density culture should be used to increase the dilution rate and activate the bacteria. In order to cultivate a large amount of antagonists, the experiment was carried out to reduce the cost of the medium material required for the cultivation by using a cheap carbon source and a nitrogen source capable of obtaining a large amount of cell mass with excellent antagonism.

Antagonists used in the experiment were isolated and identified in this experiment.B. subtilisCP220 In the nutrient agar medium, two to three times of subcultures were activated as a parent strain, and one platinum strain was cultured in 5 ml of nutrient broth and cultured. Strains were harvested and transferred to 100 ml of nutrient medium for shaking for 12 hours. In addition, after taking 1 of these and shaking culture (105rpm, Hanbaek Sci. Co. HB-201SL) for 12 hours in 100ml nutrient medium in 500ml Erlenmeyer flask, the inverter-controlled centrifuge (One-chip Microprocessor centrifuge, Hanil S750-4B) was used. After washing the cells three times with physiological saline (NaCl, 0.85) (3,000g, 10min, 4 ℃) supernatant was removed and only the cells were collected. Temperature control of the culture was controlled by a thermostat manufactured by the Organic Method Research Society, the pH of the culture was adjusted using 1N HCl and NaOH.

(1) Growth of antagonist microorganisms with temperature

In order to select the optimum growth conditions of B. subtilis CP220 in the culture medium, the growth of antagonistic bacteria was investigated in the temperature range of 25 ~ 45 ℃. Experimental results showed the best growth at 32 ℃.

(2) Growth of antagonistic microorganisms by pH

In order to investigate the optimum growth conditions of antagonists according to the pH change, the most suitable pH range was determined to be about 7 when the antagonists were cultured while changing the pH of the medium.

(3) Growth of antagonists by carbon and nitrogen sources in the medium

Selected antagonists should be cultured in large quantities to increase their activity and dilution in actual field experiments. In order to cultivate the antagonist in large quantities, the experiment was carried out to reduce the unit cost of the medium material necessary for the cultivation using a cheap carbon source and a nitrogen source capable of obtaining a large amount of cell mass with excellent antagonism. Mass growth conditions of antagonistic B. subtilis CP220 isolates identified in this study were reviewed. As a result of culturing the strain by preparing the culture medium according to the type and concentration of the carbon source and nitrogen source used in this experiment, the result showing the carbon-nitrogen source combination which was the best for the growth of the strain is shown in Table 7 below. In the case of B. subtilis CP220, the nitrogen-carbon combination with the best growth of the strain was the highest in the medium composition consisting of peptone: sugar = 5: 5 (W / W) as the carbon source. However, commercial medium was selected as soy sauce 3 and carbon commercially available sugar 5 as soy sauce and carbon source.

Growth of Antagonist Bacillus subtilis CP220 by Carbon and Nitrogen Sources Carbon Source () Nitrogen Source () Starch glucose Sugar Lactose Glycerol Galactose Soy sauce ^*) 3: 0.5 ( ^**) 34.4) 3: 5 (84.1) 3: 5 (92.3) 3: 3 (56.5) 3: 5 (32.8) 3: 1.5 (23.2) soy milk 1: 0.5 (5.4) 3:10 (12.4) 1: 5 (9.2) 2: 8 (12.7) 1: 6 (14.5) 1: 5 (12.9) Ammonium Sulfate 3: 0.5 (8.2) 1: 2.5 (15.6) 1: 2.5 (18.4) 1: 2.5 (21.4) 2: 5 (12.9) 2: 4.5 (19.8) Element 1: 0.5 (8.4) 1: 2 (1.6) 0.5: 1 (7.5) 1.2: 2 (14.8) 1.6: 3 (25.2) 1.2: 4 (11.5) Ammonium chloride 3: 0.5 (2.4) 1: 5 (18.4) 1: 5 (18.6) 1: 4.5 (19.3) 1: 4.2 (12.3) 1: 4 (15.2) Polypeptone 3: 0.5 (76.2) 1: 5 (97.0) 1:10 (85.9) 1: 8 (45.2) 1: 5 (65.3) 1: 8 (81.6) peptone 5: 1.9 (25.6) 7: 5 (80.5) 5: 5 (100) 6: 3 (76.2) 5: 1.5 (30.2) 6: 5 (83.1)

^*) Nitrogen source: carbon source ratio (mass / mass), ^**) Comparative growth degree (): Reference peptone: Sugar = 5: 5

(Cultivated at 34 ° C. for 5 days).

(Example 6) Determination of Enzyme Activity of Antagonist Microorganisms

To investigate the antagonists secreted by antagonists, chitinase and pectinase enzyme activities were measured. Chitinase is an enzyme protein found in antagonists as chitin degrading enzyme, a cell wall component of pathogenic fungi. Pectinase is an enzyme secreted by pathogenic fungi and is widely found in pathogens that cause plant disease by degrading the fruit or stem of the plant. Therefore, the two enzyme activities were measured to determine the pathogenicity of antagonists.

Pectinase enzyme activity was measured by Keen et al. For 24 hours at YC agar (ammonium sulfate 2g, magnesium sulfate, 7HOH 0.2g, casamino acid 3g, yeast extract 2g, agar 1.2, water 1 liter, pH 8.0) 32 ℃. After that, pour 5 ml of 1M CaCl ₂ into the plate medium. After being left at room temperature for 5 to 30 minutes, bacteria having a transparent ring around the flora were determined as Pectate Lyase positive. The determination of chitinase activity was incubated for 24 hours at 32 ° C. in a medium containing chitin (Yeast Nitrogen B 0.67, chitin 1.0, K ₂ HPO ₄ buffer (pH 5.5, 0.05M) 97.13, agar 1.2) The ring-producing bacterium was determined to be chitinase positive.

The isolated antagonist Bacillus subtilis CP220 showed positive chitinase activity and negative pectinase activity. Antagonist microorganisms show the activity of chitinases, and most of the pathogens are fungi. The cell wall component of the fungus is chitin. Therefore, one of the antagonist biosynthesis secreted by antagonist microorganisms is called proteinase. It is thought. Chitinase secreted by antagonist microorganisms showed antagonistic activity by dissolving and disintegrating the cell walls of the fungi.

Example 7 Changes of Enzyme Activity According to Growth Curve under Optimal Antagonistic Media Conditions

B. subtilis CP220 was identified in this experiment and the growth curves of the bacteria were determined according to the change of time under optimum growth conditions. The enzyme production of chitinase was measured on the growth curves. Enzyme activity according to the growth curve and the bacterial growth curve for antagonists is shown in FIG. Enzyme activity of antagonists showed a growth-associated pattern of growth similar to that of growth curve.

Example 8 Dilution Ratio Selection for Application of Pavement Experiment of Antagonists

(1) Selection of Dilution Ratio Using Culture Solution of Antagonist Bacillus subtilis CP220

After 48 hours incubation of Bacillus subtilis CP134, CP141 in a nutrient medium, centrifuged to remove the supernatant to obtain the supernatant, and passed through a 0.22μm nitrocellulose membrane filter (Micron Separations Inc.). Aseptic treatment. Diluting the culture of antagonists by concentration, inoculated apple pathogens to observe the proliferation of the pathogens, and even when the culture medium was diluted about 50, there was antibacterial activity against the pathogens. Therefore, some of the antagonists produced by antagonists were thought to be released into the body.

(2) Antagonist spraying concentration experiment using plant (apple) leaf extract

In order to determine the spraying concentration of antagonists under the condition of maximal packaging, extracts were obtained from plant leaves and treated with antagonists by concentration as the basic medium and inoculated with pathogens to observe the growth of pathogens. In the case of the antagonist B. subtilis CP220, 10-fold and 100-fold dilution of antagonistic bacteria was inhibited, and all of the phytopathogenic bacteria were inhibited when treated at 1,000-fold, and the dilution ratio was set to about 500-fold. 8).

Inhibitory Effect of Phytopathogenic Bacteria on Dilution Ratio of Antagonist Bacillus subtilis CP220 Antagonist pathogen dilution factor Bacillus subtilis CP220 10 100 1000 Corruption ( B. dothidea ) +++ +++ ++ Anthrax ( G. cingulata ) +++ +++ + Spot deciduous disease ( A. mali ) +++ +++ ++ White door disease ( R. necatrix ) +++ +++ ++ Ovulation ( V. ceratosperma ) +++ +++ + Gray Mold Disease ( B. cinerea) +++ +++ + Bronchopathy ( Phomopsis sp. ) +++ +++ ++ Receipt Bottle ( P. ultium ) +++ +++ + Wilting disease ( F. oxysporium ) +++ +++ ++ Root Rot Disease ( F. solani ) +++ +++ ++ Advisory disease ( H. mompa ) +++ +++ ++ Blue Mold Disease ( P.expansum ) +++ +++ +

+; 40 or less suppression, ++; 40 to 80 degree suppression, +++; 80 or more suppression

(Example 9) antagonist spraying and culture medium is decayed pathogen, anthrax pathogen, ovulation pathogen,

Effects on Blue Mold Pathogen, Root Rot Pathogen, and Gray Mold Pathogen

effect

(1) antagonist spray after pathogen inoculation

0.8 ml of the liquid cultured antagonist B. subtilis CP220 incubated for 1 day after inoculation of apple rot pathogen, anthrax pathogen, ovule bacillus, blue mold pathogen, root rot pathogen, gray mold pathogen on PDA Spray it gently. The results obtained by culturing the pathogens sprayed with antagonists in a 25 ° C. incubator for 7 days were compared with those of the non-treated control group, as shown in FIG. 2. All treated antagonists were almost completely inhibited against phytopathogens such as decayed pathogens, anthrax pathogens, ovulation pathogens, blue mold pathogens, root rot pathogens and gray mold pathogens. Therefore, the application of antagonists to the packaging gives good results.

(2) inhibitory effect of phytopathogens (corruption pathogens, anthrax pathogens, ovulation pathogens, blue mold pathogens, root rot pathogens, gray mold pathogens)

Culture medium preparation is as follows. Without-heat treat: Removed antagonists (without-heat treat): After sterilization by adding twice the amount of PDA concentrated in distilled water, centrifuged for 24 hours in a culture medium incubated in complete medium for 24 hours at 10,000rpm, pore size 0.45 mm filtered through a membrane filter (membrane filter) and mixed with the same amount of the distilled water in sterile PDA medium to make a solid medium. Heat Treatment of Antagonist-Free Cultures (121 ° C., 15 minutes): Sterilize 15 minutes at 121 ° C. for 15 minutes by mixing the centrifuged culture solution (50 ml) + distilled water (50 ml) + PDA, and make a solid medium. Inoculate the pathogen into the solid medium containing the culture solution and incubate for 7 days in a 25 ℃ incubator compared to the control group inoculated with the pathogen in PDA.

As shown in Figure 3, rot pathogens, anthrax pathogens, Bran pathogens, blue fungal pathogens, root rot pathogens, gray mold pathogens after cultivation of antagonists showed no antagonism microorganisms heat treated or annealed in the medium without antagonists This secreting substance was found to be very resistant to heat.

Example 10 Storage Stability of Antagonist Bacillus subtilis CP220

When an effective microbial preparation using antagonist Bacillus subtilis CP220 was made, we tried to obtain basic data on shelf life and storage stability by measuring the number of viable cells in liquid medium over time. After culturing the antagonist microorganisms in a commercial nutrient medium and the medium prepared in this experiment (SD medium; sugar 5, soy sauce 3, water 92) for 2 days, glycerol and molasses were added to each liquid medium and stored at 25 ° C. The viable cell count was measured over time.

Bacillus subtilis CP220 had the best shelf life when added to glycerol after incubation in SD medium or nutrient medium. After about 1 year, it maintained 10 ⁷ cell / ml. Therefore, when antagonist Bacillus subtilis CP220 was used as a microbial agent, glycerol was added to the culture medium and the shelf life of the product was set to 1 year.

Example 11 Investigation of Survival Rate of Antagonist Microorganisms in Leaves

To investigate how long antagonist Bacillus subtilis CP220 can survive on leaf after spraying on crops, kanamycine and streptomycin are used as genetic markers of antagonist Bacillus subtilis CP220. Induced strains were named Bacillus subtilis CP220 KS (Str ^R , Kan ^R ). Bacillus subtilis CP220 KS developed an antibiotic resistant mutant strain having 100 μg / L resistance to streptomycin and 50 μg / L resistance to kanamycin.

As shown in FIG. 4, about 33 days after spraying Bacillus subtilis CP220KS, about 33 microorganisms appeared to survive in the leaves. The results suggest that the microbes survive on the leaves for about 10 days. Antagonist microorganisms are sprayed every 10 days on average.

Example 12 Investigation of Antagonist Microbial Spraying Time after Pesticide Spraying for Persimmon Pesticides

Due to serious residual problems in the plant and environment of chemical pesticides, interest in environmentally-friendly farming methods using persimmon pesticides, low pollution pesticides and microbial pesticides is also increasing. In the present invention, the antagonist microorganisms sprayed when the antagonist microorganisms were sprayed after the pesticide spraying on the apple tree for the first time were investigated. Antagonists were isolated from apple leaves by spraying antagonist microorganisms 20 days after anthracol hydration and 6-12 Bordeaux pesticide spraying. Spraying antagonistic microorganisms were cultured in B. subtilis CP220KS and sprayed on the apple tree sufficiently.

Isolation of antagonist microorganisms from apple leaves was carried out using antibiotics (Kanamycin, Streptomycin) resistant strains as a genetic selection marker. Strains cultivated in the medium were isolated by spray antagonists. As shown in Figure 5, the anthracol hydration was able to survive when the antagonist B. subtilis CP220KS was sprayed 16 days after the application and 6-12 Bordeaux solution was able to grow antagonists after 18 days. In view of these results, anthracyc hydrohydrate should be sprayed with antagonistic microorganisms after 16 days, and spraying of antagonists after 18 days after 6-12 Bordeaux liquid spray is considered effective.

Example 13 Antifungal Effect of Antagonistic Bacteria in In vivo Bioassay

(1) Investigation of the inhibitory effect of rot, anthrax and blue mold

When the spore suspension of pathogens was applied to the skin without hurting the apples during storage, it was difficult to obtain experimental data because the incidence was very low and it took 20 days or more. The apples were washed with a surfactant and washed again with 10 hydrochlorite solution, and then pierced 2 mm deep with a tip of sterile toothpick around three parts of the apple's nipple and wounded. After immersing in 300 ml of the culture medium, 20 µl of spore suspension (10 ⁴ / ml) was inoculated onto the wound of the apple, and then sealed at about 28 ° C. in a sealed humid plastic chamber (57 × 36 × 21 cm ³ ). During 10 days of preservation, the incidence and inhibition of apple rot, anthrax, and blue mold were investigated. The control group was inoculated only with pathogen spores to the wound and examined under the above conditions.

After dipping the apples in the culture filtrate then the gilhanggyun icing stab sticking a toothpick into sterile dried apples at room temperature where the 20㎕ spore suspension (10 ⁴ / ml) did not smear the apple and the apple is smeared spore suspension By day 5 after inoculation, the lesions were less than 3 mm in diameter and were considered to have no progress. When the spore suspension was inoculated with a toothpick without being immersed in the antagonistic culture, 100 days of inoculation was observed at the 6th day of inoculation and the diameter of the lesion was 18 mm or more. Treatment of the culture solution showed antimicrobial activity of 50 or more at 6 days of inoculation (FIG. 6).

In the present invention, the antagonistic bacteria can be used for biological control against rot, anthrax, and blue mold disease of apples since the suppression of rot, anthrax, and blue mold disease is remarkable on the actual apple surface. In the future, it can be used as a biological control agent when the culture solution is directly sprayed on the apple tree to show the control effect of the double rot.

(Example 14) Apple bottle control using isolated antagonistic bacteria in pot cultivated apples

When the growth of the apple tree was vigorous, two spots (1 year, 2 year old) per week were wiped clean with sterile water at 20 cm intervals from the 30 cm ground of the apple, and the wooden spores were treated with a sterile dissecting scalpel and wounded. Plants inoculated with Valsa ceratosperma and Glomerella cingulata were induced for 48 hours after saturation by placing pots in the open field. Antagonist treatment method was foliar spraying for 7 days before and after pathogen treatment.

As shown in FIG. 7, the control group treated only with Bran pathogens caused the disease on the apple tree stem. However, in the apple tree treated with the antagonist B. subtilis CP220 isolated in this study, both diseases were treated before and after pathogen treatment. It did not occur. Therefore, the antagonist microorganism isolated in this study could prevent the ovulation disease, and there was some therapeutic effect when the antagonist was sprayed early after the invasion. As for anthrax, the antagonistic bacteria were foliarly sprayed every 7 days before and after pathogen treatment. In addition, the control effect of anthrax using the antagonist B. subtilis CP220 was effective only when the antagonist was sprayed in a preventive manner, as shown in FIG.

An effect of the present invention is to provide a biopesticide that biologically controls pests using bactericidal and insecticidal effective microorganisms, the present invention is to isolate the main pathogens and to develop an antagonist microorganisms as a biological control agent.

Claims (4)

Bacillus microorganism Bacillus subtilis CP220 ( Bacillus subtilis CP220: Accession No. KCTC-8831P), which has an antagonism to plant diseases isolated from nature, was grown by growth in the medium, and prepared by diluting the dilution factor about 100 to 1,000 times. Microbial preparations with plant disease antagonism The method of claim 1, wherein the pathogen of the plant applicable to the microbial agent is a rot rot (rot), anthrax, spot deciduous (spot deciduous), ovule, stem blight (dongdongjeung), sesame, beetle, wilted disease, Microbial agents characterized by one or more plant pathogens selected from among pathogenic fungi, such as aphrodisiac, gray mold, root rot, purple-wing disease, and white-wing disease. The microorganism preparation according to claim 1, wherein the strain is cultured in a medium consisting of 2.5 to 3.5 soy as a nitrogen source and 4.5 to 5.5 sugar as a carbon source. Plant disease antagonistic strain Bacillus subtilis CP220 (Accession No .: KCTC-8831P)