KR101406823B1 - Bacillus sp. CS-52 and Antifungal control against Anthracnose of Red Pepper using the same - Google Patents

Abstract

The present invention relates to a bacterial strain of Bacillus sp. CS-52 ( Bacillus sp. CS-52), a culture solution containing the Bacillus sp. CS-52, and a method for biological control of a red pepper disease using the Bacillus sp. CS-52 strain. The composition of the medium capable of maximizing the antifungal activity and the effect of increasing the growth and production of the antifungal activity and the growth of the plant by this strain is sprayed on the plant and excellent effect on the pepper anthracnose ( Colletotrichum gloesporides ) Respectively.

Description

Bacillus sp. CS-52 strain and its use in controlling pepper anthracnose {Bacillus sp. CS-52 and Antifungal control against Anthracnose of Red Pepper using the same}

The present invention relates to the control of red pepper anthracnose ( Colletotrichum gloesporides ), which is a disease of red pepper, using the isolate strain Bacillus sp. CS-52 ( Bacillus sp. CS-52). ( Bacillus sp. CS-52), a new microbial strain that inhibits anthrax that occurs most frequently during the entire growth period of pepper, .

Due to the development of fine chemical pesticides in modern society, agriculture productivity is increasing due to the development of pesticides and fertilizer, and it is solving the problems due to human food shortage. However, social interest in environmental pollution resulting from abuse and residue is greatly increased rather than the advantages of synthetic chemical pesticides and chemical fertilizers. As the quality of life of human beings is improved in accordance with the well-being age, the usage of chemical synthetic pesticides and chemical fertilizers And the demand for environmentally friendly agricultural products has been increasing. As a result, farmers' preference for organic farming has been steadily increasing.

However, it is difficult to control plant diseases when cultivating crops from field packaging to environmentally friendly farming, and it is very difficult to harvest high quality agricultural products. Studies have shown that when farming without crop protection, the amount of grooming is reduced and yields are significantly reduced. Therefore, the development of sustainable agriculture-friendly agriculture requires bio-pesticides that are effective in replacing chemical synthetic pesticides.

Biopesticides include microbial pesticides that use microorganisms to control plant diseases, insects and weeds, and biochemical pesticides that use natural products produced by plants or microorganisms. In the case of microbial pesticides, it is difficult to mass-produce and formulate the product through optimization of product, and since the storage stability of the product is low, standardization, standardization, and ease of distribution are not secured like chemical pesticides. When the microbial pesticide is used in actual packaging, the microorganism can not be effectively used for controlling the plant disease since the microorganism can propagate the microbial cells only after the microbial cell has grown to a considerable amount. In addition, since the spectrum of applied diseases is not wide, various bio-pesticides must be treated according to the types of plant hospitals generated in the crops, which realistically puts an economic burden on the farmers.

The sales of microbial pesticides are very low at 0.14% of the total pesticides, and the government and farmers are turning to the consciousness of the government and farmers as a substitute treatment of chemical synthetic pesticides through continuous research and development in universities and institutions.

In the case of foreign countries, studies on various fields such as rotation, no tillage, partial tillage, untreated direct circulation and organic livestock circulation system are being carried out mainly in Europe and Japan, and the added value of organic agriculture is relatively high. Agricultural research and application cases are increasing. In addition, Quantum4000 and Kodiak, such as Bacillus subtilis , are used for controlling horticultural crop diseases in the United States. However, biological control of pepper blight by antagonistic microorganisms has not been done yet.

However, in spite of the many research examples and global efforts, the pesticides that can effectively control various plant pathogens have not yet been established and development of new environmentally friendly biological control agents is required.

That is, an object of the present invention is to provide a novel strain exhibiting an antifungal effect against red pepper anthracnose, which is a red pepper disease.

Another object of the present invention is to provide a method for controlling a red pepper disease characterized by using the above-described novel strain and a biological pesticide preparation for controlling red pepper disease and the above-described pesticide preparation.

The above object of the present invention is achieved by a method for isolating and isolating a Bacillus sp. CS-52 strain, which exhibits antagonistic action against red pepper disease,

Preparing a microbial pesticide preparation for controlling anthrax, the new strain of Bacillus sp. CS-52 and a culture thereof as an active ingredient;

The antifungal activity was verified by using the microbial pesticide preparation, and the quality of pepper and the effect of increasing harvest were evaluated. Hereinafter, the present invention will be described in detail.

The Bacillus sp. CS-52 strain and the culture thereof according to the present invention have an excellent controlling effect on anthracnose of pepper and have an excellent effect of promoting the growth of pepper crops.

1 shows the result of 16S rRNA gene analysis of Bacillus sp. CS-52 strain of Bacillus sp. CS-52.
FIG. 2 is a systematic diagram showing the results of identification of Bacillus sp. CS-52 strain of Bacillus sp. CS-52.
3 is a morphological photograph of a Gram stain and a scanning electron microscope of a strain of Bacillus sp. CS-52 ( Bacillus sp. CS-52).
Figure 4 shows the result of antifungal activity over time after establishing the culture medium and culture conditions for the Bacillus sp. CS-52 ( Bacillus sp. CS-52) strain.
Figure 5 shows the results for the production of Siderospore and the inhibition of Spore germination of the Bacillus sp. CS-52 strain of Bacillus sp. CS-52.
Figure 6 shows the results of the production of cellulase of Bacillus sp. CS-52 ( Bacillus sp. CS-52) and IAA, a plant growth hormone.
FIG. 7 is a graph showing the results of In vitro assay results.
Fig. 8 shows the results of the change in the average production amount of the red pepper produced from the test package using the culture solution containing the Bacillus sp. CS-52 strain.
Figure 9 shows the results of the incidence of red pepper disease in the field test package.

Example  1: Isolation and selection of antagonistic bacteria

In order to select the antagonistic microorganisms that inhibit the growth of red pepper disease, seawater, shellfish samples, orchards and natural cultivated soil in the whole country were used as the root samples, suspended and diluted in sterile physiological saline, and these were suspended in MA, LB and NA agar medium And inoculated, the microorganisms were isolated by culturing at 30 DEG C for 2 days.

To isolate strains that can control anthrax, which is the cause of red pepper disease, 100ul of the culture supernatant of each strain was spotted on a paper disk, dried, and then cultured in a potato dextrose agar (PDA) The strains inhibiting anthrax mycelial growth were finally selected.

Afterwards, the taxonomic identification of antagonistic bacteria was identified through 16S rRNA sequencing, a genetic analysis method, and its genealogical and morphological characteristics were examined (FIGS. 1, 2 and 3).

Disclosure of the Invention The identified microorganism strain was deposited with KCTC 12078BP on November 21, 2011 in the international depository.

Example  2: Optimal Medium condition  Establish

The establishment of optimal culture conditions using the antagonistic bacteria will be described in detail.

The production of antagonistic substances of the Bacillus sp. CS-52 strain of Bacillus sp. CS-52 according to the culture conditions was investigated. As a result, the best strains and antagonistic substances were produced under the medium conditions described in [Table 1].

The optimum medium composition of the strain of the present invention ingredient Composition ratio (g / L) Glucose 5g K ₂ HPO ₄ 7g KH ₂ PO ₄ 2g NH ₄ NO ₃ 3g MnSO ₄ .7H ₂ O 0.1 g Yeast extract 1.5 g

As a result of the production of the antagonistic substance of Bacillus sp. CS-52 according to the present invention according to the culturing pH and temperature, the optimal production of anthracnose control agent, (Fig. 4).

Example  3: Bacillus  genus CS -52 ( Bacillus sp . CS -52) Side Rosspoa  production

The ability to produce siderospore of the strain Bacillus sp. CS-52 according to the present invention was assayed using the Chrome Azurol S liquid assay. CS-52 was cultured in the optimal culture medium. After mixing with the culture supernatant (50 mL) and CAS assay solution (Chrome Azurol S), the solution was allowed to stand for 1 to 2 minutes. The results were expressed as [(control - experimental group) / control] × 100 =% as sideropore units, as shown in FIG.

Example  4: invention Bacillus  genus CS -52 ( Bacillus sp . CS -52) and the cellulase activity of the strain Indol acetic Acid  ( IAA )

The cellulase activity of the Bacillus sp. CS-52 strain of the present invention was measured using carboxymethyl cellulose (CMC) as a substrate. After incubation at 50 ° C for 30 minutes, the cells were treated with 1.5 ml of DNS (NaOH 1%, 3,5-Dinitro Salicylic Tetrahydrate 20 (1%), 100 μl of 1% CMC, 200 μl of 0.1 M phosphate buffer (pH 7.0) and 200 μl of the culture supernatant of Bacillus sp. CS- %) Was added and boiled for 5 minutes. After stopping the reaction, the absorbance was measured at 540 nm.

IAA (Indole acetic acid) activity of the plant growth hormone CS-52 ( Bacillus sp. CS-52), which is an outbreak of Bacillus sp. CS-52, was determined by adding 0.1% L-tryptophane, a precursor of IAA, And incubated at 30 ° C for 48 hours. After culturing, the culture supernatant was centrifuged at 8,000 rpm for 10 minutes. The supernatant was mixed with Salkowski reagent (5 ml of perchloric acid, 100 ml, 0.05 M ferric choloride, 2 ml), and the reaction solution was changed to pink after 30 minutes. Absorbance was measured at a wavelength of 450 nm. The results of the experiment are shown in Fig.

Example  5: Bacillus  genus CS -52 ( Bacillus sp . CS -52) culture broth i n  vitro Protection  Verification

Pepper fruits (green pepper, red pepper) were used to verify that the pathogenic forces of the isolates of the present invention have a dragging power not only in the medium but also in the red pepper fruit. The red pepper fruit was washed once with sterilized distilled water and then once more with 3% sodium hypochlorite, wiped with 70% alcohol wet cotton, and then irradiated with UV for 10 minutes. (1 × 10 ⁶ cfu / ml) of CS-52 culture supernatant diluted with anthracnose spore suspension (1 × 10 ⁶ cfu / ml) was used to infect the pepper fruit with a disposable syringe. (Chemical pesticide + spore suspension), and cultured in a wet chamber for 6 days at 25 ° C in an incubator. After culturing for 6 days at the foot of each pathogen, History and onset of disease.

(%) Is calculated as [(incidence of antagonistic microorganism untreated incidence - incidence of antagonistic microorganism treatment) / incidence of antagonistic microorganism untreated incidence] × 100, as shown in Table 2 below.

[Note] a: DW, B: C. gloeosporioides , C: CS-52 medium + C. gloeosporioides , d: pesticide + C. gloeosporioides

In the control (control, medium treatment), it was not possible to detect any abnormalities and other abnormalities in the wound area even after 6 days. However, in case of Infection, After 6 days from onset, the symptoms began to show, and the symptoms were visible enough to be visible. However, in the case of the treatment ( Bacillus sp. CS-52), only a similar or weak infection was observed in the control (Fig. 7).

Example  6: Bacillus  genus CS -52 ( Bacillus sp . CS -52) Strain of the culture Red pepper  Control effect, Pepper  Production survey

The packaging experiment was carried out using the culture medium of the Bacillus sp. CS-52 strain of the present invention. [Table 3] shows the treatment methods from post-harvest to harvesting on February 24, 2010. The monthly production amount is shown in FIG. 8 and the final production amount is shown in FIG.

The productivity of the pepper fruit of the strain culture of the present invention date Work contents One February 24 Large intestine sowing 2 March 3-4 Pepper implant operation 3 March 14 Intestinal transplantation 4 March 30 Microbial treatment 5 April 21 Microbial treatment of potted pot (2.5ℓ / 40ℓ) 6 May 16-17 In the roots of microbes Gantry (600 ml / 20 l) And packaging  Formally 7 June 1 Microbial treatment (5 L / 500 L) 8 June 10 Microbial treatment (5 L / 500 L) and soil analysis 9 June 15 Practice Area -499 weeks, Microbial compartment -603 weeks, microbial + Compost - 684 weeks normal  Growth confirmation 10 June 28 Microbial treatment (5 L / 500 L) 11 July 12 Microbial treatment (5 L / 500 L) 12 July 29 Microbial treatment (5 L / 500 L) 13 August 1 Anthurium & Plague Incidence rate  Research 14 August 16 Anthurium & Plague Incidence rate  Research 15 August 17 Microbial treatment (5 L / 500 L) 16 August 19 Anthurium & Plague Incidence rate  Research 17 August 22 Practice Area , Microbial compartment  Pepper harvest 18 August 24 Microbial + Compost  Pepper harvest 19 August 25 Microbial treatment (5 L / 500 L) 20 August 26 Anthurium & Plague Incidence rate  Research

As described above, the present invention has an excellent effect of providing Bacillus sp. CS-52 strain and culture thereof, and is an extremely useful invention in the biocidal pesticide industry because it has an excellent effect of controlling pepper anthracnose disease and increasing the productivity of pepper crops .

Korea Biotechnology Research Institute KCTC12078BP 20111121

<110> andong national university industry-Academic cooperation foundation <120> Bacillus sp. CS-52 and Antifungal control against Anthracnose of          Red Pepper using the same <130> P5067 <140> 10-2012-0001180 <141> 2012-01-04 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1379 <212> RNA <213> Bacillus sp. CS-52 <400> 1 tgcagtcgag cggacagatg ggagcttgct ccctgatgtt agcggcggac gggtgagtaa 60 cacgtgggta acctgcctgt aagactggga taactccggg aaaccggggc taataccgga 120 tggttgttg accggatggt tcagacataa aaggtggctt cggctacgac ttacagatgg 180 acccgcggcg cattagctag ttggtgaggt aacggctcac caaggcgacg atgcgtagcc 240 gacctgagag ggtgatcggc cacactggga ctgagacacg gcccagactc ctacgggagg 300 cagcagtagg gaatcttccg caatggacga aagtctgacg gagcaacgcc gcgtgagtga 360 tgaaggtttt cggatcgtaa agctctgttg ttagggaaga acaagtgccg ttcaaatagg 420 gcggcacctt gacggtacct aaccagaaag ccacggctaa ctacgtgcca gcagccgcgg 480 taatacgtag gtgggaagcg ttgtccggaa ttattgggcg taaagggctc gcaggcggtt 540 tcttaagtct gatgtgaaag cccccggctc aaccggggag ggtcattgga aactggggaa 600 cttgagtgca gaagaggaga gtggaattcc acgtgtagcg gtgaaatgcg tagagatgtg 660 gaggaacacc agtggcgaag gcgactctct ggtctgtaac tgacgctgag gagcgaaagc 720 gtggggagcg aacaggatta gataccctgg tagtccacgc cgtaaacgat gagtgctaag 780 tgttaggggg tttccgcccc ttagtgctgc agctaacgca ttaagcactc cgcctgggga 840 gtacggtcgc aagactgaaa ctcaaaggaa ttgacggggg cccgcacaag cggtggagca 900 tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt cttgacatcc tctgacaatc 960 ctagagatag gacgtcccct tcgggggcag agtgacaggt ggtgcatggt tgtcgtcagc 1020 tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttgat ctagttgcca 1080 gcancagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140 gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1200 gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1260 tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320 gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacaccc 1379

Claims (8)

( Bacillus sp. CS-52) strain (KCTC12078BP) having a cellulase and indoleacetic acid (IAA) activity and having a gene sequence shown in SEQ ID NO: 1. A microbial preparation for the control of an anthrax ancillary anthrax of pepper characterized by comprising the Bacillus genus CS-52 strain (KCTC12078BP) of claim 1 as an active ingredient. delete delete delete A method for controlling an anthrax anemia of pepper characterized by directly treating a microorganism preparation containing the Bacillus sp. CS-52 strain (KCTC12078BP) according to claim 2 to the environment or plant in which the pepper grows. A method for growing a red pepper fruit which is characterized in that a microorganism preparation containing the Bacillus sp. CS-52 strain (KCTC12078BP) described in claim 2 is directly treated with an environment or a plant in which the pepper grows.

delete

Images