KR20130096870A - Novel antifungal bacterium bacillus methylotrophicus lks26 - Google Patents

Abstract

The present invention relates to a pesticide using Bacillus methylotrophicus ( LKS26 ) having antifungal activity, and in detail, plant pathogenic fungi Mucor spp. (Rotosis), Pyricularia oryzae (rice fever) , Pythium ultimum (wearing) bottles), Rhizoctonia solani (rice mungobyeong), Botryoshaeria dothidea (apples rot), Bipolaris sorokniana (kkaessi blotch), Botrytis cinerea (gray mold), Colletotrichum gloeosporioides (red pepper anthracnose), Pyricularia grisea (rice blast), Mycosphaerella melonis ( Specifically , watermelon vine blight disease, Phytophthora capsici , Alternaria solani (tomato crustacean), Fusarium oxysporum (coccal disease, root disease) or Sclerotinia sclerotiorum (mycotic disease) and Candida albicans (candidiasis). Bio-matrix for controlling pathogenic fungi containing the optimal culture method and the antifungal activity spectrum trix) and its use, and can be usefully used as a multifunctional pesticide with environmental conservation and purification.
The present invention relates to a novel Bacillus methyllotrophicus LKS26 having the function of antifungal activity (AF ⁺ ), and to inhibiting the growth of phytopathogenic fungi by treating the strain with crops and cultivated soil.
Bacillus methylotrophicus LKS26 of the present invention inhibits the growth of phytopathogenic fungi in crops and soil.

Description

Novel antifungal bacterium Bacillus methylotrophicus LKS26, a novel microbial bacterium with antifungal activity

The present invention is a novel strain Bacillus methyltropycus ( Bacillus) having antifungal activity methylotrophicus ) LKS26, and this strain relates to the treatment of phytopathogenic fungi by treating the crops and crop cultivation soil.

In order to control pests, weeds, and pathogenic microorganisms of crops, microbial pesticides are products that are isolated and selected from nature or modified according to their use, and the compounds secreted by microorganisms.

  An essential element in the development of microbial pesticides is to select or modify useful microorganisms having excellent antagonism, and to mass produce the selected microorganisms or drugs secreted by the microorganisms and to efficiently deliver them to the crops for use.

Recently, research and development of microbial pesticides using microorganisms has been actively conducted. Korean patent application 1990-0017551 (a new Bacillus subtilis subspecies and the use of the antifungal substance KRF-001 complex produced therefrom), Bacillus subtilis subspecies cryptiensis (producing antifungal oligopeptide-based substances) subtilis subsp . krictiensis ), but the antifungal activity spectrum has a narrow problem.

  In Korean patent application 1993-0022037 (Method for producing microbial insecticide using bioencapsulation), a natural gel matrix rich in water is mixed with water to prepare a protein-rich natural gel matrix, and after autoclaving, Bacillus terringiensis spores are mixed. Thereafter, there is a method of preparing a bioencapsulated microbial insecticide by drying, but has a narrow antifungal activity spectrum and a complicated composition and composition of the biomatrix.

  Korean Patent Application No. 1998-027468 (Improved Coating Pesticide Matrix, Preparation Method and Composition Containing It) relates to a coated pesticide matrix comprising pesticides, polymers, plasticizers, UV protection agents, activity enhancers, and suspending agents. It is known, but the antifungal activity spectrum is narrow and the effect is limited.

As the Green Round Convention on Environmental Protection and Ecosystem Conservation becomes a subject of international concern, the abuse of excessive pesticides is a serious problem and the development of biocide for plant control that replaces it competes internationally. It is emerging as a fierce core field. In order to overcome the environmental pollution problem caused by existing pesticides, it is very urgent to select antagonistic microorganisms that are easy to produce, store and apply and effectively inhibit pathogens and to separate biologically active substances. In particular, antifungal active microorganisms and substances are less toxic to mammals than conventional pesticides, no harm to plants, excellent in activity and less residual, and do not affect rotation, and should not contaminate groundwater, soil and river water.

An object of the present invention is trophy kusu (Bacillus of the Bacillus methyl novel with the function of antifungal activity (AF ⁺⁾ methylotrophicus ) LKS26 and a novel strain relating to the inhibition of the growth of phytopathogenic fungi by treating the strain with crops and cultivated soil.

The present invention is a novel strain Bacillus methyltropycus ( Bacillus) having antifungal activity methylotrophicus ) LKS26 is a multifunctional pesticide with environmental preservation and purification, and can be used for environmentally friendly soil improver, composting, and environmental purifier.

In addition, the fungal control biomatrices of the present invention using the microorganisms growing in our country economical replacement effect is high and has a high performance can significantly reduce the agricultural workforce.

1 is a photograph showing the antifungal activity of Bacillus methylotrophicus LKS26 of the present invention
Figure 2 is a diagram showing the phylogenetic root of Bacillus methylotrophicus LKS26 of the present invention
Figure 3 is Bacillus methylotropycus ( Bacillus of the present invention) methylotrophicus ) Preparation of Biomatrices Using LKS26

In order to achieve the above object, the present invention provides a microorganism having antifungal activity specific to plant pathogenic fungi.

The present invention also provides a bio-matrix for controlling plant pathogenic fungi containing the microorganism. Specifically, the bio matrix for controlling plant pathogenic fungi is composed of plant resources polymer materials, biopolymer synthetic biogels, biogels, and surfactants in addition to microorganisms.

Hereinafter, the present invention will be described in more detail.

The present invention screens a variety of environmentally friendly microorganisms with antifungal activity against pathogenic fungi.

The invention 16S Lai hanba comparing homology bojom DNA analysis, investigating the biological properties, been identified as a novel strain of the trophy kusu (Bacillus methylotrophicus) of the Bacillus methyl, trophy kusu (Bacillus them to Bacillus methyl methylotrophicus ) was named LKS26, and the strain was deposited with KCTC 12084BP on November 23, 2011, at the Korea Institute of Bioscience and Biotechnology, an international microbial deposit institution for patent application.

Bacillus methylotropycus ( Bacillus) selected in the present invention Methylotrophicus ) LKS26 was inoculated with fungi-coated PDA (Difco Co.) medium using a paper disc (diameter 6 mm) and inoculated with cell culture medium cultured in nutrient medium and incubated for 2 to 3 days at 30 ° C for phytopathogenic fungi. Antifungal activity was investigated. Bacillus Methyltrophycus Methylotrophicus ) LKS26 was tested for antifungal activity spectrum, and the antifungal activity spectrum was analyzed for a large number of plant pathogenic fungi.

In the above pathogenic fungi are Mucor spp. Pyricularia oryzae (rice blast), Pythium ultimum (loose disease), Rhizoctonia solani (riceworm), Botryoshaeria dothidea (apple rot), Bipolaris sorokniana (Sesame Seeds ), Botrytis cinerea (ash fungus), Colletotrichum gloeosporioides (pepper anthrax), Pyricularia grisea (rice fever), Mycosphaerella melonis (watermelon vine blight), Phytophthora capsici ( fermenter ), Alternaria solani (tomato round disease), Fusarium Plant pathogenic fungi and Candida , including oxysporum (locopathy, root disease) or Sclerotinia sclerotiorum (mycobacterium disease) animal pathogenic fungi such as albicans (candidiasis) and the like.

 The antifungal active bacterium of the present invention can be used as an active ingredient such as soil improving agent, composting agent, foliar spray or irrigation spray as an environmentally friendly multifunctional antifungal agent useful for environmental conservation and purification.

In addition, the antifungal active strain of the present invention is investigated by measuring the seed germination rate does not affect the growth of the plant. The antifungal active bacteria have not been shown to inhibit plant seed germination at all, which is useful not only as a microbial pesticide but also as a seed coating.

The present invention also provides a bio matrix for controlling pathogenic fungi formed by appropriately mixing a plant resource polymer material, a microgel-derived biopolymer, a cell of a biopolymer-synthetic microorganism, and the like.

At this time, the plant material polymer materials include spinach powder, cabbage powder, raw brown rice, acorn jelly powder, alpha cone, seaweed powder, fresh vinegar powder, mugwort powder, raw sugar powder, kelp powder, green bean powder, mar powder, red bean powder, chestnut powder, Wheat flour, pine needle black bean powder or carrot powder is used, and biopolymer is mucopolysaccharide (mucopolysaccharide) or poly-beta-hydroxyalkanoate, and the microorganism is nitrogen-fixed bacterium Rhizobium. loti , Rhizobium meliloti , Rhizobium japonicum Or cyanobacteria or the like.

In addition to the above composition, the biomatrix may include vermiculite, mud, alginic acid, chitin, oyster shell, fruit shell dried powder, corn stalks, chaff, sawdust, and other powders, various extracts, peptone, tryptone, sugars, inorganic salts, vitamins, It may further comprise an amino acid or flavonoid (flavonoid) and the like.

Prabonoids in the biomatrix are used as antifungal activity enhancers (Letters in Applied Microbiology. 26: 363-366, 1998).

In addition, the biomatrix uses a coating aggregate which allows the antifungal active bacteria to adhere well to the plant leaves or to be degraded in the soil. Specifically, as a coating aggregate of antifungal active bacteria, it is preferable to use a bioflocculants released by cyanobacteria into the aquatic environment, and particularly Anabaena. sp. PC-1 and Anabaena sp. Use of the N-1444 strain is advantageous for obtaining extracellular aggregates with strong aggregation activity.

The bio-matrix has excellent survival time, survival germination rate, antifungal activity, and antifungal substance generation ability of antifungal active bacteria, and is applied to all antifungal active bacteria as microbial pesticide, encapsulating material of microbial preparation, microbial carrier and seed coating agent. It can be usefully used. In addition, the bio-matrix may be used for soil improving agent, composting agent, foliar spray or irrigation spray.

The antifungal compound of the present invention may be prepared by a method known in the pharmaceutical field for use as a pesticide, and may be a carrier, excipient, forming agent, diluent, or the like, by itself or a pharmaceutically acceptable carrier. It can be prepared and used in various formulations by mixing.

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

However, the embodiments are illustrative of the present invention, and the content of the present invention is not limited to the examples.

Example 1 Selection of Strains

To search for strains from kimchi, the cells were cultured and isolated in Lactobacillus MRS medium and Trypton East Extract medium (TYE).

In addition, the isolated microorganisms were inoculated with a cell culture medium incubated in a nutrient medium using a paper disk (diameter 6 mm) in a PDA (Difco Co.) medium on which phytopathogenic fungi were smeared and incubated at 30 ° C. for 2-3 days. Antifungal activity against pathogenic fungi was investigated.

<Example 2> Investigation of genetic similarity between the nucleotide sequence of the isolated strain and the existing strain

Genomic DNA of the strain selected in Example 1 was isolated using CTAB solution.

16S ribosomal DNA (rDNA) was amplified using PCR, cloned, and its nucleotide sequence was determined.

The nucleotide sequence of the determined 16S rDNA was compared with the strains registered in the NCBI gene bank to investigate the phylogenetic tree and genetic similarity and the results are shown in FIG. 3.

As shown in Figure 3, the selected strain was shown as a new strain of Bacillus methylotrophicus ( Bacillus methylotrophicus ).

The present inventors named the strain Bacillus methylotrophicus LKS26 and deposited it on November 23, 2011 to the Korea Institute of Bioscience and Biotechnology Resource Center (Accession No .: KCTC 12084BP).

Example 3 Investigation of the Effect of Microorganisms on Plant Seed Germination Rate

It is very important to measure the seed germination rate against antifungal active bacteria, even if the antifungal active strains have good antifungal activity, so if they inhibit the growth of plants, they will not be valuable as antifungal active strains. The germination rate of non-muddy green zucchini seeds showed a germination rate of 100%, and similar to that of the reference group in the case of Oriental Bell Pepper Glory.

As such, the antifungal active bacteria do not inhibit the seed germination rate of the plant, and thus, they may be very useful for seed storage and preservation when encapsulated at the seed level.

< Example  4> Preparation of Biomatrices Using Antifungal Active Bacteria

To produce antifungal physiologically active substances while maintaining them in a natural environmental ecosystem or in plants, it is necessary to maintain nutrient supply (nutrients that can be used in the short and medium to long term), protect the antifungal bacteria, increase germination rates, and maintain long-term growth. need. Therefore, the bio-matrix (bio-matrix) having such a function was to be developed.

Specifically, about 20 kinds of biomatrices were prepared by appropriately mixing plant resource polymer material (biogel), microbial source biopolymer (microgel), biopolymer synthetic microorganism cells or other materials. At this time, the plant material polymer materials include spinach powder, cabbage powder, raw brown rice, acorn jelly powder, alpha cone, seaweed powder, fresh vinegar powder, mugwort powder, raw sugar powder, kelp powder, green bean powder, mar powder, red bean powder, chestnut powder, Uses wheat flour, pine needle black bean powder, carrot powder, etc.In addition to other materials, vermiculite, mud, alginic acid, chitin, oyster shell, fruit shell dry powder, corn stalk, rice husk, sawdust burned powder, etc., various extracts, peptone, Tryptone, sugars, inorganic salts, vitamins, amino acids or flavonoids (flavonoid) and the like were added as appropriate. In addition, mucopolysaccharide (MPS) or poly-beta-hydroxyalkanoate is used as a biopolymer of microbial origin, and mucopolysaccharide is a nitrogen-fixed bacterium, Rhizobium. loti , Rhizobium meliloti, Rhizobium japonicum Or those derived from cyanobacteria were used.

< Example  5> Bacteria adsorption Augmented  Screening of Biodegradable Coating Agglomerates

In order for the microorganism of the present invention to be used as a fungicide for controlling plant pathogenic fungi, first of all, the coating antagonist is required not only to maintain antagonistic activity even at room temperature for a long time, but also to adhere well to plant leaves or to decompose well in soil. The present invention extracted and characterized the bioflocculants released by cyanobacteria into the aquatic environment with the antifungal active bacteria obtained above. Anabaena selected from several species of cyanobacteria by kaolin clay analysis and Alcian blue binding assay sp. PC-1 and Anabaena sp. The N-1444 strain was found to secrete abundantly extracellular aggregates with strong coagulation activity. To understand the properties of this aggregate, Anabaena sp. As a result of investigating the growth curve and kaolin aggregation activity for each week while culturing PC-1 for 6 weeks, the coagulation activity was the strongest at 4 weeks until the growth was stagnant, and the hot phenol from the strain cultured for 4 weeks The coagulant was extracted using, and partially purified using CTAB and ethanol precipitation, followed by pure purification using Sephacryl S-200 column chromatography. These aggregates are macromolecular polysaccharides, consisting of 63% of the total molecular weight of neutral sugar, 5% of uronic acids, 12% of protein, but keto acids, hexosamines, Fatty acids were almost absent.

In addition, the purified agglomerates have a strong affinity for the cationic dye, Alcian blue, and thus are considered polyanionic / sulfated polysaccharides, and their coagulation activity is stronger under strong acidic conditions ( acid-dependent), coagulation activity increased by 40% even after heating at 100 ^o C for 7 min. In addition, when the salt or metal such as NaCl, NaNO ₃ , MgSO ₄ , ZnSO ₄ is added, the activity is increased, and activated carbon, silica, aluminum oxide, bentonite, chitin, chitosan, powdered agar, cellulose, DEAE, Dowex in solution -50W, some aggregation with organic / inorganic materials such as Sephadex.

< Example  6> Selection of adjuvant of antifungal active substance

In order to investigate the possibility of synergistic effect of antifungal activity when the compound of flavonoid family, which is a natural product distributed in plants rather than microorganisms, has the potential for synergistic effect of antifungal activity, or a substance such as a metal component having excellent antifungal activity but toxic to chevron, In order to examine the effect on fungal growth through interaction, the inhibitory effect of fungal growth by the interaction of zinc with quercetin and copper with quercetin was observed. This confirmed that flavonoids can be used as an antifungal activity enhancer (Letters in Applied Microbiology. 26: 363-366, 1998).

< Example  7> Preparation of Biomatrix Pesticides Containing Antifungal Active Bacteria

Manufacturing process

After sterilizing the prepared bio matrix, the antifungal active bacterium cooled and concentrated at about 50 ° C. is first coated with mucopolysaccharide (MPS) secreted by nitrogen-fixed bacteria, and mixed with the bio matrix. The number of cells per gram of the polymer material was prepared to about 10 ⁸ -10 ⁹ , and then uniformly mixed in a homogenizer to uniformly fix microorganisms evenly in the membrane. This fixed microorganism, the bio matrix composite of the mucopolysaccharide and the polymer was dried to prepare a powder of the appropriate size.

< Example  8> Investigation of Stability and Performance of Microbial Pesticides

Stability of Microbial Pesticides

It is important that the microbial pesticides have a medicinal life of at least 1 to 2 years. Therefore, the microbial pesticides were left for 0 months, 3 months, 6 months, 9 months, and 12 months, and the number of living microorganisms regenerated was investigated. Specifically, the concentration of the antifungal active bacteria was set in the experimental section, each sample was plated on a NA plate medium and incubated at 30 ° C for 24 hours, and the stability was measured by comparing the number of microorganisms initially put and the number of regenerated microorganisms. It was. As a result, compared with the control group, the encapsulated and dried pesticides were stable without any change during the 12-month storage period, although somewhat different depending on the strain.

In order to analyze the antifungal activity of the regenerated antifungal active bacteria, AF experiments were carried out by randomly selecting 50 regenerated AF ⁺ bacteria, and all of them possess AF ⁺ antagonism. It was confirmed to remain as it is.

Investigation of Leaf Surface and Soil Survival Rate of Antifungal Active Bacteria in Encapsulated Microbial Pesticides

Bacillus Methyltrophycus methylotrophicus ) LKS26 strains showed 1 ~ 2 weeks survival rate in cucumber leaf surface, cucumber root soil, and general soil, which was increased by 2 ~ 20 times compared to the initial survival rate.

Investigation of Spraying Activity of Microbial Pesticides

Bacillus Methyltrophycus Methylotrophicus ) LKS26 was encapsulated and sprayed compared to the control drug Fluazianam (200 ppm), and the control effect on cucumber gray mold was higher ( Bacillus methylotrophicus LKS26: 81%, Fluazianam: 74%).

Investigation of Germination Rate of Coated Seeds of Microbial Pesticides

Bacillus Methyltrophycus methylotrophicus ) When LKS26 was encapsulated and coated with 100 seeds of cucumber, tomato, and pepper, the germination rate of the coated seeds was not different from that of the untreated group, but rather increased.

UV resistance investigation of microbial pesticides

In Bacillus was methyl 5 hours of exposure to the sun trophy kusu (Bacillus methylotrophicus ) LKS26 strain did not reduce the survival rate by simply adding a bulking agent. Bacillus Methyltrophycus Methylotrophicus ) LKS26 strains slightly increased the number of viable cells when formulated with an appropriate combination of extender, MPS, UV blocker, wakame powder, vegetable powder, nutrients.

Claims (3)

A microorganism Bacillus methyl having antifungal activity against pathogenic fungi trophy kusu (Bacillus methylotrophicus ) LKS26 (Accession No .: 12084BP) The method of claim 1, wherein the plant pathogenic fungi include Mucor spp. (Corruption), Pyricularia oryzae (rice fever), Pythium ultimum (wort disease), Rhizoctonia solani (rice rot), Botryoshaeria dothidea (apple rot), Bipolaris sorokniana (seed seed pattern) Disease), Botrytis cinerea (ash fungus), Colletotrichum gloeosporioides (chili anthracnose), Pyricularia grisea (rice fever), Mycosphaerella melonis (watermelon vine disease), Phytophthora capsici (chile disease), Alternaria solani Fusarium oxysporum (wearing bottle, Root Rot) or Sclerotinia sclerotiorum (gyunhaekbyeong) and trophy kusu (Bacillus of the Bacillus is selected from methyl, it characterized in that the animal pathogenic fungi candida albicans (candida) methylotrophicus ) LKS26 The method of claim 1, wherein Bacillus methylotropycus ( Bacillus) methylotrophicus ) LKS26 is effective for microbial pesticides, seed coating agents, microbial pesticides for controlling fungal diseases of mushrooms, additives for the production of non-sterile media for mushroom cultivation, biosoils, microbial nutrients, soil improvers, composting agents, foliar sprays or irrigation sprays. Bacillus methylotrophicus LKS26 , characterized by being used as a component

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