KR101508728B1 - New microorganism bacillus amyloliquefaciens naas-1, and microbial agent and biopesticide containing the same - Google Patents

Abstract

The present invention relates to a novel microorganism Bacillus amyloliquefaciens NAAS-1, a microorganism preparation containing the microorganism, and a microbial pesticide.
The new microorganism Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) of the present invention is represented by SEQ ID NO: 1 and is characterized by inhibiting the growth of pathogenic bacteria caused by Chinese cabbage blight.
According to the present invention, by providing a new microorganism Bacillus amyloliquefaciens NAAS-1 having excellent control effect against Chinese cabbage blight, a microorganism preparation containing them and microbial pesticides, it is possible to grow crops by suppressing the occurrence of Chinese cabbage blight disease So that the number of crops can be increased.

Description

[0001] The present invention relates to a novel microorganism Bacillus amyloliquefaciens NAAS-1, a microorganism preparation containing the microorganism Bacillus amyloliquefaciens NAAS-1, and a microbial agent containing the microorganism Bacillus amyloliquefaciens NAAS-

The present invention relates to a novel microorganism Bacillus amyloliquefaciens NAAS-1, a microorganism preparation containing the microorganism, and a microbial pesticide containing the same. More particularly, the present invention relates to a method for isolating and isolating a strain capable of inhibiting the growth of pathogenic microbes, And a microbial agent and a microbial pesticide containing the strain as an active ingredient.

Bacterial soft rot disease in plants is the most problematic disease in vegetable cultivation. It is found in almost 50 kinds of plants, including vegetables, fruit vegetables, potatoes and bulbs, which form a fresh storage organ. During the cultivation period Of course, it is a disease that causes great damage during transportation or storage of harvest.

Especially in Chinese cabbage, it occurs in leaves, stems and roots. It develops from left to right in the wound starting from scratch. Finally, the tissue turns into a cream, which emits a bad smell and causes some or all of the cabbage to die.

Chinese cabbage rot was caused by Chinese cabbage every year and it caused great damage, and it is one of the biggest limit factors in the production of cabbage in summer.

Currently, streptomycin and oxolinic acid are registered as pesticides for the control of this disease. However, when the density of pathogenic bacteria increases in the cabbage and cabbage packages in summer, Is very low.

In addition, antibiotics or chemicals used for controlling plant diseases have been devoted to the development of environmentally friendly biocides since the 1980s, mainly in developed countries, due to the generation of antibiotic resistance in agricultural ecosystems and pollution and residual toxicity to the environment.

Particularly, such a pesticide-based control method is environmentally safe, has almost no toxicity to human body, and generally uses microorganisms that are already widely available in the natural world, minimizing the resistance to use of pesticides and variations in natural ecosystems There are advantages to be able to.

A related prior art is Korean Patent Publication No. 10-2002-0064386 (published on Aug. 09, 2002, entitled: Novel Bacillus sp. Strain having antimicrobial activity against a bacteriostatic pathogenic pathogen and a method for controlling bacterial blight ) And Korean Patent No. 10-0580336 (registered on May 09, 2006, name: New Bacillus sp. Microorganism having the effect of controlling Chinese cabbage blight).

DISCLOSURE OF THE INVENTION An object of the present invention is to identify and isolate Bacillus amyloliquefaciens NAAS-1, a novel strain that exhibits antimicrobial activity against pathogenic bacteria caused by Chinese cabbage blight, thereby enabling the growth of crops, and a microorganism preparation containing the strain and microbial pesticides .

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not intended to limit the invention to the particular embodiments that are described. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are not restrictive of the invention, There will be.

Novel microorganism of the present invention for achieving the above object Bacillus amyl quinone Lowry Pacific Enschede (Bacillus amyloliquefaciens ) NAAS-1 (KACC 91751P) is represented by SEQ ID NO: 1 and is characterized by inhibiting the growth of pathogenic bacteria caused by Chinese cabbage blight.

The novel microorganism Bacillus amyl Lori rake Pacifico Enschede (Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) is isolated from soil and is used as an active ingredient of any one of microbial pesticides, seed coating agents, soil improving agents, composting homework, leaf foliar spraying agents, or traditional spraying agents.

The pathogenic bacteria of the Chinese cabbage rot disease include Pectobacterium carotovorum subsp . Carotovorum , Pack tobak Te Solarium Lee Shipman Pedy (Pectobacterium cypripedii), Pseudomonas deep corruption in Ostrava (Pseudomonas viridiflava ) , Pseudomonas sp. marginalis pv . marginalis ) Or more.

Further, the microorganism preparation of the present invention is the novel microorganism Bacillus amyl quinone Lowry Pacific Enschede (Bacillus amyloliquefaciens ) NAAS-1 (KACC 91751P) or a culture solution thereof as an active ingredient.

The culture broth contains soluble starch as a carbon source, yeast extract powder as a nitrogen source, and salt as an inorganic salt. Preferably, the soluble starch is contained in an amount of 0.5 to 3.5% by weight based on the weight of the culture solution, And the salt is contained in an amount of 0.001 to 0.05% by weight relative to the weight of the culture liquid. More preferably, the soluble starch is 2% by weight based on the weight of the culture liquid, 5% by weight, and the salt is 0.01% by weight relative to the weight of the culture solution.

Further, microbial agricultural chemicals of the present invention is the novel microorganism Bacillus amyl quinone Lowry Pacific Enschede (Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) or a culture thereof as an active ingredient. The microbial pesticide is characterized in that the microorganism pesticide is sprayed on the soil or in the soil to control the Chinese cabbage blight.

The present invention provides a novel microorganism Bacillus amyloliquefaciens NAAS-1 having excellent control effect against Chinese cabbage blight, microorganism preparation containing it, and microbial pesticide containing it, thereby enabling growth of crops to be inhibited It is possible to increase the yield of crops.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the identification of a novel microorganism Bacillus amyloliquefaciens NAAS-1 strain of the present invention. FIG.

The terms, techniques, and the like described in this specification are used in the meaning commonly used in the technical field to which the present invention belongs, unless otherwise specified.

The new microorganism Bacillus amyloliquefaciens NAAS-1 strain of the present invention is represented by SEQ ID NO: 1, and is isolated from soil and exhibits antimicrobial activity against the causative pathogens of cabbage root rot by the following Examples .

At this time, the isolated strain is a staphylococcus, the form of spore is oval, is located at both ends of the cell, and the sporangia is not inflated. In the nutrient broth, the cultivation was good for 1-2 days at 30 ° C, no pellicle was formed, no precipitate of the cells was observed, the growth temperature range was 20 to 50 ° C, the growth pH range was 4 to 10, It can grow up to 8% salt concentration. And catalase positive, oxidase negative, and propionate availability, hydrolysis of starch but no blood hemolysis

Microbial pesticides and microbial pesticides for inhibiting the development of Chinese cabbage smut disease containing the microorganism Bacillus amyloliquefaciens NAAS-1 strain of the present invention or a culture thereof may be provided.

Particularly, in the culture solution, soluble starch as a carbon source, yeast extract powder as a nitrogen source, and salt as an inorganic salt are contained in the culture solution so that the microorganism Bacillus amyloliquefaciens NAAS-1 of the present invention can be cultured well Preferably, 0.5 to 3.5% by weight of the soluble starch, 2.5 to 7.5% by weight of the yeast extract powder and 0.001 to 0.05% by weight of the salt, based on the weight of the culture, The optimal conditions for the growth of the strain are that the soluble starch is 2 wt% based on the weight of the culture, the yeast extract powder is 5 wt% based on the weight of the culture, and the salt is in a weight ratio 0.01% by weight.

When the soluble starch is contained in an amount of less than 0.5% by weight, the energy-producing ingredient is too small to slow the growth rate of the strain. When the soluble starch is contained in an amount of more than 3.5% by weight, Thereby slowing the growth rate of the strain.

In addition, when the yeast extract powder is contained in an amount of less than 2.5% by weight, the growth and reproduction of the strain are slowed down, and the specificity of the catalyst to be selectively catalyzed is deteriorated. When the yeast extract powder is more than 7.5% by weight, excessive catalyst is added, There is a possibility that proper mixing is not achieved and at the same time strain of the strain is caused.

When the salt is contained in an amount of less than 0.001% by weight, the effect of a central role in metabolism is deteriorated, so that the growth rate of the strain is lowered. When the salt is contained in an amount exceeding 0.05% by weight, But rather slows the growth rate of the strain.

Such microbial agents and microbial pesticides include a culture solution prepared by culturing the above-mentioned strains under the above-mentioned culture conditions, and are excellent in survival survival period, survival rate, antimicrobial activity retention, Antimicrobial microbial pesticides, seed coating agents, microbial nutrients, soil amendments, composting homework, foliar spraying agents or cross-linked spraying agents.

The present invention will be described in more detail with reference to the following examples and experimental examples, but the scope of protection is not limited to the following examples and experimental examples.

Example 1 Isolation and Identification of the Novel Antagonistic Microorganism of the Present Invention

1) Isolation of soil microorganisms

The novel microorganism of the present invention was isolated from the soil of the Chinese cabbage cultivation area (Gangwon Province Hwacheon Organic Cabbage Cultivator) and identified as shown in Fig.

2) Selection of the microorganism strain of the present invention

The soil collected from the organic cabbage cultivation area of Hwacheon was suspended in distilled water and platinum, and then plated on NB plate medium (juice 0.3%, peptone 0.5%, agar 1.2%) and cultured at 30 ° C. for 1-2 days to isolate the strain .

The strains isolated in 10 ml of nutrient broth (NB medium) were inoculated once with platinum and cultured at 30 ° C for 76 hours with shaking.

The culture was centrifuged by applying centrifugal force at 12,000 × g (gravity acceleration) for 20 minutes and cultured by paper disc method using the bacteria of the genus Pactobacterium carotovorum subsp. Carotovorum KACC 10057 as test specimens The antimicrobial activity of the supernatant was measured.

Among the isolated strains, the strains with the best antimicrobial activity were selected and identified as similar to Bacillus subtilis, but showed differences in growth salt utilization and cyto - rate utilization.

However, 16s rDNA analysis showed 100% agreement with Bacillus amyloliquefaciens Bacillus amyloliquefaciens NAAS-1, which was deposited with the Korean Agricultural Microbiology Center (KACC) on Nov. 1, 2012, and received the accession number KACC 91751P.

3) Identification of the Bacillus subtilis strain of the present invention

The identification of the Bacillus NAAS-1 strain of the present invention was carried out according to the identification method which is commonly used in the technical field of the present invention, and the results are shown in the following Tables 1, 2 and 3.

1. Experimental Method

DNA samples were prepared for isolating strains using 16s rDNA PCR.

In order to prepare DNA from the colonies, 100 占 퐇 of sterilized distilled water was placed in a microtubule and the cells collected from the colonies were suspended. This suspension is heat-treated at 95 占 폚 for 15 minutes. The supernatant was then used as a sample DNA solution by gently centrifuging.

Below 16s rDNA region amplification and nucleotide sequence information analysis were performed by a general manual (published by TAKARA).

2. Experimental results

Morphological characteristics of the Bacillus sp. NAAS-1 strain of the present invention shape Sweet bacillus Cell size 1.6 占 3.8 占 퐉 motility + Gram stain + Spore formation Oval Spore position Both ends

'+' Indicates positive, '-' indicates negative

As shown in Table 1, the Bacillus sp. NAAS-1 strain of the present invention is a monocytic bacillus, the spore is oval in shape, and is located at both ends of the cell and the sporangia are not inflated.

The culture characteristics of the NASS-1 strain of the genus Bacillus of the present invention Colony type circle Colony surface lubricity Colony corners wave Colony elimination protrusion Colony Cloudiness Opaque surface Colony color cream color Colony gloss Not shining

As shown in the above Table 2, the growth was good in the nutrient liquid medium at 30 ° C for 1 to 2 days, and no pellicle was formed, and no precipitate of cells was observed.

The physiological characteristics of the Bacillus sp. NAAS-1 strain of the present invention Growth temperature 20 to 50 ° C Growth pH range 4 to 10 Growth NaCl <8% Catalase + Oxidase - Urea + Lipase _ ? -galactosidase + Arginine dihydrolazate - Phenylalanine diamine - Hydrolysis: starch
Casein
cellulose
Esculin +
+
-
+ Indole formation - Formation of H ₂ S in TSI agar medium - Levan formation from sucrose + NH ₃ formation from peptone + Formation of NH ₃ from arginine + Gelatin liquefaction + Use site rate - Propionate use + Methyl red test + Nitrogen reduction + Denitrification + Milk reaction: solidified
Peptonization -
+ Blood hemolysis - O-F test Fermentation Tyrosine degradation -

'+' Indicates positive, '-' indicates negative

As shown in Table 3, the growth temperature range of the Bacillus sp. NAAS-1 strain of the present invention ranged from 20 to 50 ° C, the growth pH range was from 4 to 10, and the growth was possible up to a salt concentration of 8% by weight. And catalase positive, oxidase negative and propionate availability, hydrolysis of starch but no blood hemolysis.

&Lt; Experimental Example 1 >

1) Pyrolysis caused by cabbage blight

From the Agricultural Microbial Bank (KACC), as shown in Table 4 below, four species of cabbage rotivirus inducers were distributed. These strains were well grown and were selected as test bacteria after the activity test.

KACC No. Scientific name Incubation temperature Growth 10057 Pectobacterium carotovorum subs sp carotovorum 28 ℃ ++ 10063 Pectobacterium cypripedii 28 ℃ ++ 10387 Pseudomonas viridi flava 28 ℃ ++ 10466 Pseudomonas marginalis pv . marginalis 28 ℃ ++

2) Antimicrobial activity experiment

In order to confirm the antimicrobial activity of the new microorganism of the present invention against the microorganism causing the Chinese cabbage blight, trytic soy broth (20 ml) in a 100 ml Erlenmeyer flask ), Plated on a shaker at a temperature of 30 DEG C for 48 hours, and the strain that reached the stopper was used as the seed of the present invention.

The results are shown in Table 5 below.

 The novel microorganism Bacillus amyloliquefaciens NAAS-1 KACC No. 10057 10063 10387 10406 24hr - - - - 48hr + - - - 72hr ++ _ + + 96hr +++ + ++ + 120hr ++ + + +

 -: 8 mm +; 8 to 10 mm ++; 15mm +++: 18 ~ 20mm

mm: degree of antibacterial zone (inhibition zone size)

As shown in Table 5 above, the activity of the new microorganism Bacillus amyloliquefaciens NAAS-1 of the present invention was assayed to show antimicrobial activity against all the Chinese cabbage rot causing bacteria. 10057 strain of the present invention. The KACC 10057 strain is a representative pathogen occurring in cabbage and crops.

<Experimental Example 2> Inhibition of Chinese cabbage pathogens Bacillus amyloliquefaciens NAAS-1

The large-scale culture conditions of the novel microorganism Bacillus amyloliquefaciens NAAS-1 strain of the present invention were examined and examined as shown in Table 6 below.

Chemical condition Carbon source: soluble starch 2.0 wt%
Nitrogen source: 2.5 wt% of yeast extract powder
Inorganic salts: 0.01% by weight of salt Physical condition Temperature: 35 ℃
Stirring speed: 120 rpm
Aeration: 0.2L / min
pH (acidity): 7.0

<Experimental Example 3> Confirmation of antimicrobial activity against rot fungi according to medium composition

The antimicrobial activity of the rot fungi was confirmed by varying the composition of the medium, as shown in Table 7 below. At this time, the antimicrobial activity was measured after culturing for 72 hours.

Control
(General commercial medium for bacteria: TSB medium) Treatment
(Test result medium: ATBN-1 medium) Casein 17 wt%
Dextrose 2.5 wt%
Salt 5.0 wt%
2.5 weight% of dipotassium phosphate Carbon source: soluble starch 2.0 wt%
Nitrogen source: 2.5 wt% of yeast extract powder
Inorganic salts: 0.01% by weight of salt 14mm 24mm

Suppression area size indication: mm (paper disk diameter size 8mm)

As shown in Table 7, the novel microorganism Bacillus amyloliquefaciens NAAS-1 of the present invention exhibits higher antibacterial activity in ATBN-1 medium containing soluble starch as a carbon source, yeast extract powder as a nitrogen source and salt as an inorganic salt Respectively.

Accordingly, a novel microorganism Bacillus amyloliquefaciens NAAS-1 having excellent control effect against Chinese cabbage blight is provided, a microorganism preparation containing it and a microbial pesticide are provided, and it is possible to grow a crop by suppressing the occurrence of cabbage blight, It is possible to increase the yield.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. You can implement the examples. The scope of the present invention is defined by the appended claims, and all differences within the scope of the claims are to be construed as being included in the present invention.

National Institute of Agricultural Science KACC91751P 20121101

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> NEW MICROORGANISM BACILLUS AMYLOLIQUEFACIENS NAAS-1, AND          MICROBIAL AGENT AND BIOPESTICIDE CONTAINING THE SAME <130> P2012-0168 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1438 <212> DNA <213> Bacillus amyloliquefaciens NAAS-1 <400> 1 gctcaggacg aacgctggcg gcgtgcctaa tacatgcaag tcgagcggac agatgggagc 60 ttgctccctg atgttagcgg cggacgggtg agtaacacgt gggtaacctg cctgtaagac 120 tgggataact ccgggaaacc ggggctaata ccggatggtt gtttgaaccg catggttcag 180 acataaaagg tggcttcggc taccacttac agatggaccc gcggcgcatt agctagttgg 240 tgaggtaacg gctcaccaag gcgacgatgc gtagccgacc tgagagggtg atcggccaca 300 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 360 ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggttttcgga tcgtaaagct 420 ctgttgttag ggaagaacaa gtgccgttca aatagggcgg caccttgacg gtacctaacc 480 agaaagccac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540 ccggaattat tgggcgtaaa gggctcgcag gcggtttctt aagtctgatg tgaaagcccc 600 cggctcaacc ggggagggtc attggaaact ggggaacttg agtgcagaag aggagagtgg 660 aattccacgt gtagcggtga aatgcgtaga gatgtggagg aacaccagtg gcgaaggcga 720 ctctctggtc tgtaactgac gctgaggagc gaaagcgtgg ggagcgaaca ggattagata 780 ccctggtagt ccacgccgta aacgatgagt gctaagtgtt agggggtttc cgccccttag 840 tgctgcagct aacgcattaa gcactccgcc tggggagtac ggtcgcaaga ctgaaactca 900 aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggtcttg acatcctctg acaatcctag agataggacg tccccttcgg 1020 gggcagagtg acaggtggtg catggttgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgcaac gagcgcaacc cttgatctta gttgccagca ttcagttggg cactctaagg 1140 tgactgccgg tgacaaaccg gaggaaggtg gggatgacgt caaatcatca tgccccttat 1200 gacctgggct acacacgtgc tacaatggac agaacaaagg gcagcgaaac cgcgaggtta 1260 agccaatccc acaaatctgt tctcagttcg gatcgcagtc tgcaactcga ctgcgtgaag 1320 ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc gggccttgta 1380 cacaccgccc gtcacaccac gagagtttgt aacacccgaa gtcggtgagg taaccttt 1438

Claims (11)

Pseudomonas viridiflava , Pseudomonas marginalis pv. Marginalis , having the nucleotide sequence shown in SEQ ID NO: 1 and being isolated from the cabbage cultivation soil, are selected from the group consisting of Pectobacterium cypripedii , Pseudomonas viridiflava , Which inhibits the growth of pathogenic microorganisms of the Chinese cabbage,
New microbial Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) strain.
delete delete delete The method according to claim 1,
The novel microorganism Bacillus amyloliquefaciens NAAS-1 strain
A microbial pesticide, a seed coating agent, a soil improving agent, a compostant homework agent, a foliar spraying agent, or a root spraying agent,
Lori novel microorganism Bacillus amyl quinone Pacifico Enschede (Bacillus amyloliquefaciens ) NAAS-1 (KACC 91751P).
A culture medium containing the novel microorganism Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) according to any one of claims 1 to 5 as an active ingredient,
0.5 to 3.5% by weight of soluble starch, nitrogen source, 2.5 to 7.5% by weight of the yeast extract powder as a carbon source, 0.001 to 0.05% by weight of salt of the culture medium as the inorganic salt, &Lt; / RTI &gt;
Microorganism preparation inhibiting the occurrence of Chinese cabbage blight.
delete delete The method according to claim 6,
Wherein the soluble starch is 2 wt% based on the weight of the culture, the yeast extract powder is 5 wt% based on the weight of the culture, and the salt is 0.01 wt%
Microorganism preparation inhibiting the occurrence of Chinese cabbage blight.
Preparing a culture solution containing the microorganism Bacillus amyloliquefaciens NAAS-1 (KACC 91751P) strain of any one of claims 1 to 5;
0.5 to 3.5% by weight of soluble starch and nitrogen source based on the weight of the culture medium, 2.5 to 7.5% by weight of yeast extract powder and inorganic salts based on the weight of the culture liquid, 0.001 to 0.05% by weight, Of salt to prepare a culture medium for mass production;
Culturing the culture medium for mass production under the conditions of a temperature of 35 DEG C, a stirring speed of 120 rpm, aeration amount of 0.2 L / min, and a pH of 7.0 to mass-produce the new microorganism
Method for mass production of novel microorganism Bacillus amyloliquefaciens NAAS-1 (KACC 91751P). delete

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