KR100890013B1 - Novel strain Bacillus subtilis ＫＫＧ-1 and microbial preparations and microbial pesticides containing the same - Google Patents

Abstract

The present invention is a novel strain Bacillus subtilis ( Bacillus) to promote crop growth while having a wide range of antifungal activity subtilis ) relates to KKG-1 and microbial agents and microbial pesticides containing the same as an active ingredient.

Bacillus subtilis KKG-1 is a microorganism having an antifungal activity spectrum against P. phytophthora capsici in the control of pepper blight , and microorganisms and pesticides for controlling pathogenic fungi containing the same are provided. .

Bacillus subtilis KKG-1 of the present invention not only promotes the growth of crops in addition to antifungal activity, but also has the ability to decompose organic matter, pesticide resistance and pesticide, and thus can be usefully used as a multifunctional pesticide with environmental conservation and purification.

Description

Bacillus subtilis KKG-1 and microbial agent and biopesticide containing the same

The present invention is a novel strain Bacillus subtilis KKG-1 and an active ingredient which promotes crop growth while having antibacterial activity against the pepper bacterium Phytophthora capsici in the control of pepper disease. The present invention relates to a microbial agent and a microbial pesticide containing the same, and more particularly, to a microorganism and a microbial pesticide containing and identifying a strain having both antagonism and crop growth promotion at the same time.

An essential element in the development of microbial preparations and pesticides is to select or modify useful microorganisms with excellent antagonism, 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.

In this regard, the prior art is Bacillus subtilis subspecies producing antifungal oligopeptide-based material in Korean Patent Application No. 1990-0017551 (new Bacillus subtilis subspecies and the use of the antifungal substance KFR-001 complex produced therefrom). Although it is related to Bacilus subtilis subsp. Krictiensis , there is a problem of narrow antifungal activity spectrum.

In addition, Korean Patent Application No. 1998-0062437 (microorganisms having antifungal activity against pathogenic fungi, antifungal microbial pesticides containing the same and a method for preparing the same), Bacilius subtilis var. Amyloliquefaciens ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and the like showing a microorganism that exhibits antifungal activity and a biomatrices using the same, but the composition of the biomatrix and a narrow antifungal activity spectrum has a problem.

On the other hand, the red pepper which is the object of the present invention is an important crop of tropical and subtropical regions, a crop that is consumed not only in Korea but also worldwide, and has high nutritional and economic value to farmers and consumers. In recent years, pepper cultivation has worsened the physicochemical properties of soil and increased the resistance of plant pathogens due to the accumulation of toxic compounds, making it difficult to control late blight.

One of the most serious diseases in peppers is caused by the fungal phytophthora capsici , which causes root and crown rot, pepper leaves, stems, and fruit blight (Mao et al. 1998). ). Although the disease caused by the phytopsora has been reported as the most important cause of reducing pepper production, there is no effective control method. The reason is that the follicles of Phytophthora capsici are dry tolerant and resistant to low temperatures and other extreme adverse conditions, allowing them to survive in the soil for years without host plants.

As a control method, avoiding the series and raising the mockup to improve drainage helps manage the disease, but it is not a perfect control method. In addition, good results can be obtained by using fungicides such as methyl bromide, but plant toxins and residual pesticides are emerging as major problems that threaten environmental pollution and human health. Moreover, residual pesticides in the soil affect the density and useful plant growth of useful microorganisms, leading to a decrease in crop yield.

For these reasons, there is a need to provide pesticides based on multifunctional strains that can antagonize pepper disease and help crop growth. In particular, the demand for eco-friendly agricultural products has soared recently, reducing the use of chemical pesticides and increasing the use of eco-friendly products. However, most of them are imported from foreign countries and rarely need to provide new microorganisms. have.

The present invention has been made to solve the above problems, isolates and identifies novel strains that promote crop growth with antimicrobial activity against the phytophthora capsisai, a pathogen of red pepper disease, microbial agent containing the strain And to provide pesticides.

The above object of the present invention, in addition to the action of inhibiting the growth of phytophthora capsisai, a causative agent of pepper disease, the bacterium Bacillus subtilis KKG, which promotes growth by reducing the stress of the plant (ACC deaminase) secreted by antagonists -1 was isolated, and the antibacterial activity against the isolated strain Bacillus subtilis KKG-1 against phytopsora capsaisa and the substances secreted by antagonists reduced the stress of plants to promote growth, and then Bacillus Microbial preparations containing pestilus KKG-1 and pesticides were prepared to confirm the inhibitory effect of pepper blight.

According to one aspect of the invention,

It provides a Bacillus subtilis KKG-1 strain having an antimicrobial activity against the pepper germ bacterium Phytoprosora capsaici.

According to the second aspect of the present invention,

Provided is a microbial agent for inhibiting the development of pepper blight, comprising the strain according to the first aspect or a culture medium thereof as an active ingredient.

According to the third aspect of the present invention,

Provided is a microorganism pesticide for inhibiting the development of pepper blight, comprising the strain according to the first aspect or the culture medium as an active ingredient.

Hereinafter, the present invention will be described in detail.

Bacillus subtilis KKG-1 of the present invention, as well as having antimicrobial activity against the bacteriophage bacterium Phytoprosora capsaish as well as promoting the growth of the crop was identified through the following examples (see FIGS. 1 and 3). .

At this time, the strain isolated from the present invention forms endogenous spores as shown in Table 1 below, and the size of the grams (+) is 0.6-0.8 x 2-5 μm and the colony form on the medium was round and viscous milky white. (Table 2). In addition, as a result of using Sherlock system Version 3.00 (Microbial Id Inc.) for fatty acid methyl ester analysis of the cell membrane of the isolated strain, the unsaturated fatty acid characteristic of Bacillus sp. Api 50 CHB kit was commissioned by Micro ID of Biotechnology Co., Ltd., and was identified as Bacillus subtilus KKG-1 by showing 84.7% similarity with Bacillus subtilus. On April 24, 2007, Korea Research Institute of Bioscience and Biotechnology The gene bank was deposited with the accession number KCTC11124BP.

In addition, the growth promoting action of the crop was identified through the following examples that the substance secreted by the antagonist (ACC deaminase) is obtained by reducing the stress of the plant (see FIGS. 2 and 4).

In addition, the present invention by mixing the above-described Bacillus subtilis KKG-1 as follows to make a microbial preparation, microbial pesticides.

<Microorganisms>

1st process 2nd process 3rd process Surfactants Nutrients 1 Nutrients 2 4th process 5th process 6th process Cryoprotectants stabilizator antiseptic 7th process 8th process 9th process Thickener 1 Thickener 2 Culture

Using the stirrer in the same manner as described above mix well the components including the culture medium.

<Microbial Pesticides>

1st process 2nd process 3rd process Surfactants Thickener Culture

Surfactant + thickener; It mix | blends as 50% and a culture solution 50%.

Such microbial preparations and microbial pesticides are excellent in the maintenance of survival time, survival germination rate, antimicrobial activity, and ability to produce antimicrobial substances of active bacteria, so antimicrobial microbial pesticides, seed coatings, microbial nutrients, soil improving agents, composting agents, foliar sprays or irrigation It may be used for a spraying agent.

Furthermore, the microbial pesticides for control made of the Silus subtilis KKG-1 according to the present invention has the advantage of having a high economical replacement effect and high performance using microorganisms native to Korea.

As described above, the Bacillus subtilis KKG-1 of the present invention not only promotes crop growth in addition to antifungal activity, but also has an organic decomposition ability, pesticide resistance and pesticide decomposition ability, which is useful as a multifunctional pesticide with environmental conservation and purification. Can be used.

Hereinafter, the specific configuration of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example  1: present invention New  Isolation and Identification of Antagonist Microorganisms

Isolation of Soil Microorganisms

The novel strain of the present invention is isolated and identified through the following process from the pepper root of the root region of red pepper plantation (Deokjin-dong 1ga pepper packaging Deokjin-gu Jeonju-si, Jeollabuk-do).

Isolation of Antagonists

It was isolated from the root zone and separated using King's medium B medium or nutrient agar medium. Pepper root was added to 200 ml of phosphate buffer, stirred and washed with water. After diluting 10 times, 100 μl was dispensed into KMB medium and incubated at 28 ° C. for 2 days.

All isolates were separated according to colony size and morphology and stored in KMB / NA slope medium at 4 ° C.

Identification of antagonists

Identification method extracts DNA from antagonists, amplifies 16s rDNA using 27F (5'-AGAGTTTGATCATGGCTCAG-3 '), 1492R (5'-GGATACCTTGTTACGACTT-3') primer, and compares homology of nucleotide sequences with blast. , Antagonists were identified. The obtained result is as follows.

<16S rDNA 29f-primer sequence analysis result>

CGCTGGCGGCGTGCCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCACACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA

<16S rDNA 1492r-primer sequence analysis result>

ATCATCTGTCCCACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGT

TACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTC

The homology of the nucleotide sequences was analyzed using the blast of the gene bank. As a result, NC_000964 Bacillus subtilis subsp. subtilis str. The nucleotide sequence homology of 168 and 725 bp was identified as a subtype of Bacillus subtilis.

The isolated strain formed endogenous spores as shown in Table 1 below, and the size was 0.6-0.8 x 2-5 μm as a gram (+) bacterium, and the colonies on the medium had rounded and viscous milky white (Table 2). . In addition, fatty acid methyl in the cell membrane of the isolate

Using Sherlock system Version 3.00 (Microbial Id Inc.) for ester analysis, the unsaturated fatty acid, which is the characteristic of Bacillus sp., Was particularly high (63%). Note) The micro ID was identified as Bacillus subtilus KKG-1, showing 84.7% similarity to Bacillus subtilis, and deposited on April 24, 2007 under the deposit number KCTC11124BP to the Gene Bank of Korea Biotechnology Research Institute. It was.

Mycological Properties of Bacillus subtilis KKG-1 of the Invention characteristic Bacillus subtilus KKG-1 Bacillus subtilus shape Simple Simple Gram reaction + + Cell size 0.7 × 0.9 × 2-8 μm 0.8 × 0.7 × 2-5㎛ motility 70-90% 90-100% Growth temperature (high) 40-50 ℃ 35-50 ℃ Growth temperature (low) 5-20 ℃ 5-20 ℃

In addition, in order to know the optimal pH in the potato liquid medium of Bacillus subtilis KKG-1 of the present invention, the strain was inoculated into a 500 mL Erlenmeyer flask containing 250 mL of PD broth, and then shaken at 30 ° C. using a shaker (100 rpm). After culturing for 24 hours at the temperature of the absorbance of the culture was measured. The results are shown in Table 2 below, and it was found that cell growth was most active at pH 6.

Cell growth according to pH of Bacillus subtilis KKG-1 of the present invention pH 3 4 5 6 7 8 Cell Growth (Abs 660nm) 0.28 0.32 1.3 1.54 1.23 1.02

Example  2: biological control experiment ( in in vitro )

Antagonistic bacteria were shaken in each liquid culture at 150 rpm for 48 hours at room temperature. Bacterial cells were centrifuged at 6,000 rpm for 15 minutes and then resuspended in phosphate buffer. Bacterial concentration was used as the inoculum when the spectrophotometer was 10 ⁸ cells / ml at the absorbance 595nm.

mold

Storage cultured antagonistic fungi were incubated in PDA medium for 25 ° C./3 days. If the size of mycelium grows about 5 mm long in the first medium, it is transferred to the second PDA medium, and then healed and incubated at 25 ° C. for 7 days to prepare a spore suspension. The prepared suspension was added to sterile distilled water in colony using a glass spreader to separate spores.

The concentration of each fungal strain is counted using a haemocytometer and diluted to 10 ³ cells / ml.

The spores of each antagonistic fungus are suspended in a 0.75% guar gum solution and the suspension is inoculated into pepper seeds. Pepper seeds from the control were soaked in sterile guar gum solution.

Double culture

Bacterial / fungal isolates were tested for the ability to inhibit the growth of P. capsici in PDA and CMA plate media by dual culture (Yoshida et al., 2001).

The mycelia of P. capsici (1 cm in size) were wound in the center of agar medium in 90 mm Petri dish and made 4 dots on the edge of the plate to inoculate the suspension of isolates and incubated for 48 hours. Mold growth inhibition was confirmed by measuring the length of the lowland.

The results obtained are shown in Table 3 below.

division Antagonism P. capsici P. citrophora P. citricola R13 ++ ++ +++ R14 ++ + + R15 ++ ++ ++ R19 + + + R26 ++ ++ ++ R34 ++ ++ +++ KKG-1 +++ +++ ++

* +: 1-5 mm area, ++: 6-10 mm area, +++: 10 mm or more area.

** The results refer to the mean value of three replicates.

Vigour index

The seeds were surface sterilized with 1% sodium hypochlorite for 30 seconds and washed several times with distilled water. Seeds were each inoculated with antagonistic microorganisms and placed on wet paper and incubated for 15 days.

Seed germination rates were recorded and the vigour index was calculated using the formulas of Abdul baki and Anderwon (1973), as shown in Equation 1 below.

Vigour index = (root length + shoot length) x germination (%).

The experiment was repeated three times and the results obtained were summarized in Table 4 below.

division Vigour index % Increase compared to control R13 630 54.4 R14 597.8 46.5 R15 621.6 52.24 R19 665 62.95 R26 631.4 54.70 R34 668.11 63.70 KKG-1 704.9 72.70 Control 408.1

* Control group: grow only with distilled water

Example  3: Detached leaves assay

This example is to evaluate the ability of antagonistic microorganisms to prevent infection of P. capsici .

The antagonistic microbial cells were suspended in distilled water and sprayed onto 5 cm long pepper leaves of the isolated plant, and wet filter paper was placed in Petri dishes and the leaves were placed thereon. A spore suspension of P. capsici containing 10 ³ strainers per ml was prepared and sprayed on the leaves with antagonistic microorganisms.

Inoculated leaves were incubated at 20 ° C. under light / dark conditions for 12 hours for 10 days. The incidence was evaluated in stages 0-4, depending on the browning part: 0: disease free, 1: 1-12%, 2: 13-25%, 3: 26-50%, 4: 51-100% .

The browning degree was repeated three times and the average value of the obtained results is summarized in Table 5 below.

Experimental Example No. division Antagonism One R13 1.8 2 R14 2 3 R15 1.5 4 R19 1.4 5 R26 1.7 6 R34 1.4 7 KKG-1 1.0 8 P. capsici 3.6

Example  4: biological control of seedlings

Surface sterilized seeds were inoculated into antagonistic microorganisms, respectively, and 200 microliters of a spore suspension of P. capsici (10 ³ scavenger / ml) was placed in a plated medium.

Incubated for 20 days in the growth phase (70% humidity, 12 hours light conditions 22 ℃, 12 hours dark conditions 20 ℃). The incidence was evaluated in stages 0-5: 0: disease free, 1: leaf slightly wilted, stem tip browning, 2: 30-50% mortality, 3: 50-70% mortality, 4: 70-90% mortality , 5: 100% all deaths.

In addition, the possibility of plant growth promoting ability of antagonistic microorganisms was measured by measuring shoot length, root length, biomass and hay quantity of the seedlings, and the results are shown in Table 4 by calculating the vigour index.

Example  5: help promote growth ACC deaminase

1-aminocyclopropane-1-carboxylate deaminase (ACCD) is secreted from the enzymes isolated from soil bacteria, and the precursor ACC of plant hormone ethylene is converted to α-ketobutyrate and ammonium. . In order to amplify the ACC deaminase gene, ACC deaminase gene was amplified and electrophoresed using sequence-specific forward (5'-ATGAACCTGCAACGATTC-3 ') and reverse (5'-TCAGCCGTCTCGGAAGAT-3') primers. The confirmed result is described in FIG.

Production Example  One: Bacillus Subtilus KKG Preparation of Microbial Agents Containing -1

<Microorganisms>

1st process 2nd process 3rd process Surfactants Nutrients 1 Nutrients 2 4th process 5th process 6th process Cryoprotectants stabilizator antiseptic 7th process 8th process 9th process Thickener 1 Thickener 2 Culture

Using the stirrer in the same manner as described above well mixed the components including the culture solution.

Production Example  2: Bacillus Subtilus KKG Preparation of microorganisms containing -1 Preparation of microorganisms containing

Bacillus subtilis KKG-1 of the present invention was cultured and concentrated. Concentrated Bacillus subtilis KKG-1 was encapsulated with mucopolysaccharide (MPS). The encapsulated Bacillus's Sup-blocks KKG-1 cells were not bio-matrix gram per 10 ⁸ - 10 were mixed to about ^9, and then mixed uniformly in a homogenizer.

The Bacillus subtilis KKG-1 and the bio matrix composite thus prepared were dried to prepare a powder of a suitable size to prepare a microbial pesticide.

<Investigation of Stability and Performance of Microbial Pesticides>

(1) Stability of Microbial Pesticides

It is important that microbial pesticides have at least 1-2 years shelf life.

The microbial pesticide prepared above was left for 0, 3, 6, 9, 12 months and then the number of living microorganisms sacrificed was investigated.

Set the experimental section of the concentrated Bacillus subtilis KKG-1, each sample was plated on a NA plate medium and incubated for 24 hours at 30 ℃, and compared with the number of microorganisms initially put and the number of regenerated microorganisms stability Was measured. As a result, the microbial pesticide of the present invention, which was encapsulated and dried compared to the control, was somewhat different but stable during the 12 month storage period.

In addition, in order to analyze the antimicrobial activity of the sacrificed Bacillus subtilis KKG-1, an antimicrobial activity (AF) experiment was performed by selecting 50 randomly regenerated bacteria. As a result, all of them retained AF + antagonism and confirmed that all of the regenerated strains retain their biological antimicrobial activity.

(2) microbial pesticides UV  Resistance survey

The UV resistance of the microbial pesticides prepared in Preparation Example was investigated. Survival of Bacillus subtilis KKG-1 in microbial pesticides exposed to sun for 5 hours was not reduced.

(3) Toxicity investigation of microbial pesticides

The microbial pesticide containing Bacillus subtilis KKG-1 of the present invention was prototyped and then orally injected into the mice with pesticides, and cytotoxicity test, skin irritation test and acute toxicity test were performed.

As a result, the microbial pesticide of the present invention showed no toxicity at all even when administered 2,000 mg / kg higher than the general pesticide injection standard, and also showed an effect of increasing weight compared to the control group.

Production Example  3: Preparation of microbial nutrients using microbial pesticides

A microbial nutrient was prepared using the microbial pesticide prepared in Preparation Example 2.

Microbial pesticides of the present invention and biopolymers (microgel), plant nutrients (nitrogen, phosphoric acid, potassium, amino acids, other minerals, etc.), electrodeposition (sticker), filler (filler), UV blocker, etc. Made microbial nutrients.

In the case of microbial nutrients, when sprayed on plants, not only the growth of the crops but also the bacillus subtilis KKG-1 of the present invention was excellent in controlling plant diseases.

The present invention has been made to solve the above problems, isolates and identifies a novel multifunctional strain that promotes crop growth with antimicrobial activity against the phytophthora capsisai, a pathogen of red pepper disease, containing the strain Microbial agents and pesticides may be provided. In addition, the microbial pesticide for control made of Bacillus subtilis KKG-1 of the present invention has the advantage of having a high economical replacement effect and high performance using microorganisms native to Korea.

1 is a view showing the preventive effect of phytopathogens among the multifunctional effect according to an embodiment of the present invention specifically (a) is Phytophthora phytopathogens A photograph showing the antagonism of the capsici (A: antagonist strain, B: phytopathogen), (b) the antagonistic photos (C: treatment, D: no treatment) during the seedling period of the treatment, and (c) the pretreated microorganism Means the picture (C: no treatment, D: treatment) preventing phytopathogen infection from the leaves.

Figure 2 is a view showing the effect of promoting crop growth of the multi-functional effect according to another embodiment of the present invention, (a) is about 150% more developed roots after germination (A: untreated, B (B) is the difference in growth (A: no treatment, B: treatment) when treated at the seedling season, and (c) growth and growth test of leaves and stems (A: no treatment, B). (Process) The result is shown.

Figure 3 is an electrophoresis picture of 16S rNDA of antagonists according to an embodiment of the present invention.

Figure 4 is an electrophoretic picture of the ACC gene causing growth promotion according to an embodiment of the present invention.

Claims (4)

It has antimicrobial activity against Phytophthora capsici and expresses 1-aminocyclopropane-1-carboxylate deaminase (ACCD) gene. Bacillus subtilis KKG-1 (KCTC11124BP) strain that promotes the growth of crops. A microbial agent for inhibiting the development of red pepper disease, comprising the strain of claim 1 or a culture medium thereof as an active ingredient. A microbial pesticide for inhibiting the development of pepper blight, comprising the strain of claim 1 or a culture medium thereof as an active ingredient. delete

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