KR20060021162A - Antifungal plant growth promoting rhizobacterium, bacillus amyloliquefaciens mj-3 and microbial bed soil using hereof - Google Patents

Abstract

The present invention is to provide a microbial soil using an antifungal crop growth promoting fungi, and in detail, to generate a chitinase having a control activity against pathogens acting in the vicinity of pathogenic fungi causing disease in plants. In addition, to provide a novel microbial Bacillus amyloliquefaciens MJ-3 strain having a purpose of promoting the growth of crops, and further formulated it microorganisms to provide microbial soil mixed in various clay raw materials The purpose.

Bacillus amyloliquefaciens MJ-3 strains according to the present invention, which can be effectively utilized as an environmentally friendly control method, produce a variety of plant pathogens because they produce chitinases that have control activities against root diseases of various plants. Not only can control the growth of the fungus, but also has the advantage of promoting the growth of crops, it is expected to contribute greatly to the bio-pesticide industry and horticulture agriculture by applying to horticulture seedling soil.

Microbial Topic, Bacillus amyloliquefaciens, chitinase

Description

Antifungal Plant Growth Promoting Rhizobacterium, Bacillus amyloliquefaciens MJ-3 and Microbial Bed Soil Using hereof}

The present invention relates to microbial soil using antifungal crop growth promoting fungi, and in particular, to produce a chitinase having a control against pathogenic fungi causing disease in plants and to promote the growth of crops Bacillus amylory The present invention relates to a microorganism soil identified by identifying a strain of Bacillus amyloliquefaciens MJ-3, and microbial formulation thereof into various soil raw materials.

As a kind of artificial seedling material that creates the seedling environment of crops, it is a good physicochemical adsorbent and high quality so that it can support the plant for the planned period from planting or transplanting to planting and grow without pest or physiological disorder. It refers to agricultural materials made by mixing organic matter and various essential inorganic nutrients necessary for growth in a certain ratio, and are generally used under various names such as cultured soil, mixed soil, soil, soil, mixed media, artificial soil, and the like.

There are four conditions for horticulture seedling tops, the first of which is that the quality of the topsoil material is uniform and the bobbin power is moderately high. In other words, the topsoil should not only be properly contained and maintained in the nutrients necessary for crops, but also be excessively excessive, such as the temporary overproduction of nutrients. Secondly, the topsoil should not change the physicochemical properties. The proper acidity and proper specific gravity should be maintained according to the cultivation method, and it should be able to supply the necessary oxygen to the roots and perform the gas exchange function to smoothly discharge carbon dioxide. It should be able to protect and support crops. Third, the topsoil should be sterilized in a proper way, free of harmful germs and pests in seedlings, and free of weed seeds. Lastly, it should be highly reproducible to produce evenly small seedlings, and it is good soil to be able to maintain optimal breathability for root respiration.

Since ancient times, the top soil is made by using organic materials such as leaflets, rice straw, barley straw, livestock powder, lime, and oil on the soil or paddy soil, and adjusting the pH and stacking fermentation promoting material for 6 months or more and then ripening it 2-3 times. I have used it. In recent years, the company has been manufacturing topsoil with the convenience and excellent effects of topsoil.The main materials are organic materials such as peat moss, coco peat, rice hulls, hultan, lobule, bark, cooked sawdust, and pearlite, vermiculite, granulated rock wool, granular Two or more kinds of inorganic materials such as styrofoam, zeolite, masato and bulk loam are mixed, respectively, and mixed with modifiers such as lime, bioactive agent, acid and hygroscopic agent.

The problem of top soil is small container in seedling, even though top soil with a lot of air content is inevitable after proper growth is inevitable. The coating appears severely, the fertilizer dissolves due to excessive irrigation, and the sediment appears quickly, and the soil soil with very low porosity is used in the container of small cells, resulting in deterioration of growth, and the acidity and water conductivity of the irrigation water. It is high, so salts accumulate in the soil and inhibit the growth of seedlings, and it causes respiratory failure of the roots due to over-humidity by using airless port, and random mixing of other soil material to inhibit pathogen contamination and soil uniformity. Keep the bottom of the product in close contact with the ground to prevent pathogen contamination, soil aeration, moldability, etc. It has a problem.

Until now, organic synthetic pesticides, which have been used since 1902 together with mechanical civilization and chemical fertilizers, have played a key role in solving human food problems.However, the use of organic synthetic pesticides reduces human immunity due to soil and water pollution. In addition to the reduction of their preservation capacity, they also lead to environmental pollution and ecosystem imbalances that threaten humanity as a whole. Therefore, recently, biological protection measures to solve the food problem while reducing the burden on the ecosystem as a means to replace the chemical pesticides are actively researched, and the interest in the control of biopesticides or microorganisms is increasing.

In order to control plant diseases microbiologically, microorganisms that function effectively as a microbial fertilizer by mixing various useful microorganisms such as nitrogen-fixed microorganisms, organic decomposition microorganisms, and photosynthetic microorganisms, mainly using antagonistic bacteria with antagonism to various pathogens. It is a good useful microorganism only if it lives in the root zone of the target crop.

Biocontrol methods using antagonistic microorganisms against various phytopathogenic fungi, such as plant root disease, can be classified into three types. First, fungal envelope hydrolase chitinase, β-1,3-glucanase, and recently reported plant chitinase from Streptomyces, Antrobacterium, Trichoderma, Myroteccomb, etc. Control method by lytic action to decompose cell pathogens of phytopathogens, and secondly, to inhibit the growth of phytopathogens directly by antifungal antibiotics produced by Streptomyces kasugaiensis, Bacillus subtilis, Pseudomonas, etc. Antibiotic control, and third is a method using a competitive antagonism that inhibits the growth of phytopathogens by siderophore, an iron-specific binding substance secreted by the root Pseudomonas genus.

In recent years, as the application of chemical fertilizers and pesticides and the pollution of agricultural production environment due to livestock wastes gradually increase, it is required to develop environmentally-friendly microorganisms and to develop environmentally friendly agriculture. It is necessary to promote the growth of plants through the use, supply various fertilizers, and increase the biological control ability against diseases such as root infectious diseases.

In order to control plant diseases microbiologically, microorganisms that function effectively as a microbial fertilizer by mixing various useful microorganisms such as nitrogen-fixed microorganisms, organic decomposition microorganisms, and photosynthetic microorganisms, mainly using antagonistic bacteria with antagonism to various pathogens. It may be a good useful microorganism only if it lives in the root zone of the target crop, but most commercial microorganisms do not survive in the root zone of the actual crop or resist the root secretion of the crop, resulting in a significant decrease in rooting ability. There is no big effect of useful microorganisms. In particular, it is important to understand the environment of the subterranean rhizosphere because mozzarlock disease that causes the greatest damage to young seedlings is caused by pathogens inhabiting the roots of the subterranean root.                         

Therefore, microorganisms useful for microbial preparations and soils are applied by separating and selecting the root zone microorganisms that are antagonistic against Mozalok disease and promoting the growth of crops among indigenous or predominant root zone microbes living in the root zone of existing crops. It is available.

Therefore, the present inventors have started the present invention for plant control using microorganisms antagonizing plant root disease, and identified a new microorganism Bacillus amyloliquifaciens MJ-3, by producing a microbial soil using the antifungal crop Promoting growth microbial tops have been provided.

In order to achieve the above object, in the present invention, the common fungus (Alternaria solani), gray mold fungus (Botrytis cinerea), Fusarium oxysporium (Pusarium oxysporium), which is common in eggplant crops (tomato, pepper, eggplant, etc.) It was an object of the present invention to provide a useful microorganism, Bacillus amyloliquefaciens MJ-3 strain with high antagonistic activity to various plant pathogenic fungi such as Phytophthora capsici and Sclerotinia sclerotiorum. To provide topsoil.

The amyloliquifaciens MJ-3 strain of the present invention was identified using Burgess identification, identification using the Sherlock system, 16S rDNA sequencing, and deposited with accession number KCTC 10628BP.

Alternaria solani, Botrytis cinerea, Fusarium oxysporium, Phytophthora capsici, Sclerotinia sclerotiorum common to eggplant crops (tomatoes, peppers, eggplants, etc.) The Bacillus amyloliquefaciens MJ-3 strain, which antagonizes various phytopathogenic fungi, etc., is a gene bank (Korea Collection for Biotechnology Research Institute of Korea Institute of Science and Technology), an international depository institution under the Budapest Treaty on May 10, 2004. Type Cultures, Korea Research Institute of Bioscience and Biotechnology) was deposited with the accession number KCTC 10628BP.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Identification of MJ-3 Strains Antagonistic to Eggplant Pathogenic Fungi

Alternaria solani, Botrytis cinerea, Fusarium oxysporium, Phytophthora capsici, Sclerotinia sclerotiorum common to eggplant crops (tomatoes, peppers, eggplants, etc.) Identification of microbial strain MJ-3 strain having a high antagonistic effect on the back was performed by three methods.

First, the morphology, culture, physiology and biochemical characteristics of microorganisms are examined according to the procaryotes and Bergey's manual of systematic bacteriology. The Sherlock system (Microbial ID Inc., hereinafter referred to as 'MIDI') was identified as a Sherlock system (microorganism identification device, GC: HP Co., 6890 series), and the third was DNA sequencing (16S rDNA sequence). I sympathized with the law. PCR primers were used as R14 (5'-ACg ggC ggT gTg TAC-3 ': SEQ ID NO: 1) and R15 (5'-gCC AgC AgC CgC ggT A-3': SEQ ID NO: 2) Sequencing data was identified by searching for homology in a ribosomal database (http://rdp.cme.msu.edu/html/analyses.html).

The physiological and biochemical characteristics of the MJ-3 strain were examined according to the method of Burgy's Manual of Systematic Bacteriology, and they were positive in gram staining and had multiple flagella under electron microscopy. , Endogenous spores were formed. Cells are typical rods, 0.5 x 3.8 μm in size, flagella attached to the sides and catalase to produce a representative Bacillus genus. In addition, glucose was used to produce acid, hydrolyzed starch, and it was impossible to grow under anaerobic conditions or at low temperature of 3 ℃. Similar Bacillus species with these characteristics include B. subtilis, B. amyloliquefaciens, B. maroccanus, and B. pacificus. .

Cell wall fatty acid analysis of cells by MIDI system, mainly C14: 0 iso, C14: 0, C15: 0 iso, C15: 0 anteiso, C15: 0, C16: 1 w7c alcohol, C16: 0 iso, C16: 1 w11c , C16: 0, C17: 1 w10c, C17: 0 iso, C17: 0 anteiso, saturated fatty acids C15: 0 iso (29.34%), C15: 0 anteiso (39.65%), C16: 0iso (2.41% ), C16: 0 (551%), C17: 0 iso (10.14%) and C17: 0 anteiso (9.13%). As a result, Bacillus subtilis had a significance of 0.631, that is, a similarity of 61.3%, and Bacillus amyloliquefaciens had a significance of 0.625 and B. pumilus with 0.409. I had.

As a result of determining 16S rRNA sequence (590bp) and examining homology, MJ-3 strain showed high similarity with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of 99.48%, as shown in [Table 1], and Bacillus atropes. (B. atrophaeus) 99.28%, B. vallismortis (99.15) and B. subtilis (B. subtilis) showed a similarity of 98.98%, MJ-3 strain Bacillus amyloriquifaciens ( Bacillus amyloliquefaciens or Bacillus amyloliquefaciens was identified as M.

TABLE 1

Example 2 Culture and Storage Conditions of Bacillus amyloliquefaciens MJ-3 Strain

Bacillus amyloliquefaciens MJ-3 strain (KCTC 10628BP) was added to a 3 l Erlenmeyer flask with 2% glucose, 1.0% bigi flour, 1.5% yeast extract, 0.10 % Magnesium sulfate (MgSO4) or manganese chloride (MnCl2), pH 7.0, 500ml of distilled water incubated at 37 ℃ for 2 days (48 hours) and then used as a seed culture medium. The culture medium of the MJ-3 strain was added to a storage container containing 5g, 3ml, 1g, and 1g of sterilized rice bran, molasses, skim milk, and yeast extract at 121 ° C, mixed well, and frozen at -20 ° C to freeze-drier. drier) and then vacuum sealed is stored at 4 ℃.

Example 3 Characterization of Chitinase Produced by Bacillus amyloliquefaciens MJ-3 Strain

Since the MJ-3 strain produces 3.0-4.0 units per ml of the chitinase that degrades chitin in the culture, the chitinase of the strain was purified and its characteristics were investigated.

First, the MJ-3 strain was prepared in a jar fermentor incubator (based on 200 L) with 3% glucose, 1.0% bigi flour, 1% yeast extract, 0.10% MnCl2 (pH 7.0). The culture temperature was 37 ° C., and the air injection amount was 1 vvm and the stirring speed was fixed at 200 rpm, followed by incubation for 72 hours. After culture, the number of microbial cells was confirmed to contain 109-1010 as a colony forming unit (cfu) per 1ml.

Cultures of the MJ-3 strain were ammonia sulfate fractionation ((NH4) 2SO4 fractionation, 40% to 70%), affinity adsorption, ion column chromatography (DEAE-sephadex A-50 choromatography), gel filtration ( The final yield was purified by Sephadex G-200 gel filtration) to a final yield of 6.8%, enzyme inactivity of 59.32 units / mg, and purification of 20.4 times.

As a result of examining the properties of the purified enzyme, as shown in [Table 2], the molecular weight was 50kDa, and the optimum pH and temperature were 5.0 and 50 ° C. In the temperature stability study, the purified enzyme maintained the residual activity of 90% to 50 ° C but was 60 ° C. In the above, the enzyme activity was suddenly inactivated. Ca2 +, Mn2 +, Mg2 + and other ions activated about 20% or more enzymes, but Cu2 + ions inhibited about 80% enzymatic activity. SDS, p-CMB, MIA and other enzymes also inhibited enzyme activity. chitin) was 2.74 mg / ml.

TABLE 2

Example 4 Antagonistic Effect on Various Plant Pathogenic Fungi by MJ-3 Strains

Antagonistic investigation of various plant pathogenic fungi was carried out by pairing plate culture.

In other words, put a mass of pathogen cells (1cm x 1cm) grown in PDA medium (potato dextrose agar) in the center of PDA medium and inoculate 1 colony with 1 colony of MJ-3 strain at one edge and incubate at 37 ° C for 10 days, followed by pathogens. Antagonistic activity was determined by checking the inhibition zone of mycelia.

As a result, as shown in [Table 3], A. solani, B. cinerea, F. oxysporium, inversely occurring in eggplant crops (tomato, pepper, eggplant, etc.) Plant pathogenic fungi, including P. capsici, S. sclerotiorum, S. cepivorum, A. alternata, Anthrax, C. gloeosporioides, and stem blight (Phoma sp.), S. solani, S. sclerotiorum, F. oxysporium f. Sp. Niveum, and P. ultimum. .

These results confirm that the MJ-3 strain has a high antagonistic activity against fungi, such as Fusarium that the cell wall of the plant pathogenic fungus is composed of chitin.

TABLE 3

Example 5 Preparation of Microbial Agent Using MJ-3 Strain

For the formulation of microbial preparations, pulverized huntan (or ordinary charcoal) is used as the adsorbent (carrier) at about 50 mesh.The microbial culture medium is approximately 2.0 x 109 cfu per ml of cell and 1kg of charcoal powder as carrier in 1L of microbial culture medium. After mixing 1kg of rice bran (rice bran) well, incubate at 37 ℃ for 24 hours (incubate well for 3 hours in constant temperature room, etc.) and dry in dryer (less than 40 ℃) to control moisture content. (Typically 10%) to prepare a Huntan (or regular charcoal) microbial product.

Example 6 Preparation of Microbial Soil Using MJ-3 Strains

Topsoil was prepared according to the general method in the raw material mixing ratio as shown in Table 4.

That is, 41% coco peat, 28% peat moss, 16% pearlite, 11% vermiculite and 6% (w / w) of the chaff Huntan microbial agent prepared above are mixed and sprayed 12 kinds of nutrients dissolved in water. (spray) and the moisture content was adjusted to 40 ± 3% to prepare a microbial soil. 150-180 mg / L for ammonia nitrogen (NH4-N), 120-140 mg / L for nitrate nitrogen (NO3-N) and 190-280 mg / L for average phosphoric acid (Av. P2O5) were added. The 12 nutrients include 50-60ppm of potassium (K), 60-70ppm of calcium (calcium, Ca), 50-60ppm of sulfur (S), 50-60ppm of magnesium (magnesium, Mg), sodium (sodium, Na) ) <30 ppm, chlorine (chloride, Cl <<40 ppm, boron (B) <0.5 ppm, iron (Fe) <0.1 ppm, manganese (Mn) <0.5 ppm, zinc (zinc, Zn) < 0.01 ppm, copper (Cu) <0.01 ppm, molybdenum (Mo) <0.001 ppm.

TABLE 4

Example 7 Physicochemical Analysis of Microbial Soil Using MJ-3 Strains

As a result of analyzing the physicochemical properties of microbial soils, as shown in [Table 5], pH is 5.5-6.5, EC is 1.0 or less, CEC is more than 10 cmol + / ℓ, water content is 40 ± 3%, water holding capacity is 30 ± 5 ( 5 kPa), volume density of 0.3 ± 0.05 (Mg / m3), harmful components were below the standard amount. The chaff Huntan microbial agent was added 6% and the number of MJ-3 strain was more than 1.0 x 106 cfu / g.

TABLE 5

Example 8 Initial Growth Promoting Effect of Red Pepper Seedling by Red Pepper Microorganism

Initial growth promoting test of red pepper plug seedlings by microbial soil was carried out using red pepper, Capsicum annuum L., Seminis Korea Co. The effect of

In other words, the seeds were soaked in water at 17∼20 ℃ for 12 hours and germinated in a constant temperature room at 28 ～ 30 ℃ using a wet towel for 2 days, and then mixed with seedlings in the styrofoam box (20 x 30 x 15 ㎝). 50%, 30% of foliate soil, 20% of river sand), 2/3 of them, supply enough moisture, sowing, cover the sand, cover with vinyl, and cultivate in a constant temperature room at 23 ~ 27 ℃ for 30 days. Each time, it was transplanted into a 105-neck tray (using Biosoil Co., Ltd.). It was performed by dividing the prepared microorganism soil into a 105-hole tray (test zone) and a section (control zone) without the rice husk charcoal microorganism preparation in the clay production. The day temperature was 22-25 ° C and the night temperature was 16-22 ° C. The nutrient (an existing commercial fertilizer company) was sprayed at 5 days intervals and seeded according to conventional pest control methods. The seedlings were planted on November 13, 2002, and surveyed on January 17, 2003. The test plots were placed in three repetitions of the ovary method to examine the growth of plug seedlings, leaf, live weight, and root weight.

As a result, as shown in [Table 6], the height of the grass was increased by about 4.3% compared to the untreated soil at 21.20cm, and the cultivation showed no significant difference between the untreated soil and the untreated soil. Root length was about 7% longer than control, and biomass increased by 9.3%. However, the biomass of roots was 0.54 g / plant in control, but the test group increased by 25.9% to 0.68 g / plant.

TABLE 6

The specific parts of the present invention have been described in detail above, and for those skilled in the art, these specific descriptions are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention relates to a method for producing microbial clay using antifungal crop growth promoting fungi, and more particularly, to generate chitinase having a control activity against pathogenic fungi causing disease in plants and also to grow crops. It is to provide a method for producing microbial clay prepared by identifying a new Bacillus amyloliquefaciens MJ-3 strain that promotes the microorganism and incorporating it into various clay raw materials.

Effectively, the Bacillus amyloliquefaciens MJ-3 strain produces chitinase with plant disease control activity, which not only controls the growth of various plant pathogenic fungi, but also the MJ-3 strain promotes crop growth. Has an advantage.

<110> KIM, jin ho <120> Antifungal Plant Growth Promoting Rhizobacterium, Bacillus          amyloliquefaciens MJ-3 and Microbial Bed Soil Using hereof <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 1 acgggcggtg tgtac 15 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> PCR primers <400> 2 gccagcagcc gcggt 15 <210> 3 <211> 590 <212> DNA <213> Bacillus amyloliquefaciens MJ three <400> 3 gcggcgtgcc taatacatgc aagtcgagcg gacagatggg agcttgctcc ctgatgttag 60 cggcggacgg gtgagtaaca cgtgggtaac ctgcctgtaa gactgggata actccgggaa 120 accggggcta ataccggatg gttgtctgaa ccgcatggtt cagacataaa aggtggattc 180 ggctaccact tacagatgga cccgcggcgc attagctagt tggtgaggta acggctcacc 240 aaggcgacga tgcgtagccg acctgagagg gtgatcggcc acactgggac tgagacacgg 300 cccagactcc tacgggaggc agcagtaggg aatcttccgc aatggacgaa agtctgacgg 360 agcaacgccg cgtgagtgat gaaggttttc ggatcgtaaa gctctgttgt tagggaagaa 420 caagtgccgt tcaaataggg cggcaccttg acggtaccta accagaaagc cacggctaac 480 tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggaat tattgggcgt 540 aaagggctcg caggcggttt cttaagtctg atgtgaaagc ccccggctca 590  

Claims (6)

Bacillus amyloliquifaciens MJ-3 strain (Accession No. KCTC 10628BP). Nucleotide sequence of Bacillus amyloquifaciens MJ-3 strain according to SEQ ID NO: 3. A method of using Bacillus amyloliquifaciens MJ-3 strain for plant control. A method of using Bacillus amyloliquifaciens MJ-3 strain to promote the growth of plants. Microbial soil using Bacillus amyloliquifaciens MJ-3 strain. The method of claim 5, Microbial clay is characterized in that the microbial clay, characterized in that the prepared Bacillus amyloliquifaciens MJ-3 strain containing 6%.