KR101979954B1 - Bacillus amyloliquefaciens strain and microbial agent for the prevention and control in accordance with this cold-weather damage - Google Patents

Abstract

The present invention relates to a method for preventing or controlling microbial cold damage to crops. This invention relates to Bacillus amyloliquefaciens strains, and to a culture medium and a microorganism preparation therefor, particularly to a new strain of Bacillus amyloliquefaciens KACC 92117P which alleviates damage to apples in cold weather, There is an excellent effect in preventing or controlling apple cold. The present invention also provides a microorganism preparation for prevention or control of apple cold damage, which comprises the above-mentioned strain.

Description

TECHNICAL FIELD [0001] The present invention relates to a Bacillus amyloliquefaciens strain and to a microorganism preparation for prevention and control of a cold air pollution caused by the Bacillus amyloliquefaciens strain,

The present invention is a microorganism formulation comprising Pacifico Enschede (B. amyloliquefaciens) and its strain, especially apples, and cold weather and have excellent strain preventing or controlling effect against strawberry cold weather, the strains of the microorganism to the preparation according to the Bacillus amyl Riku It is about.

Bacillus (Bacillus), among the 151 kinds of current two subspecies (June 2008) exists, and the B. amyloliquefaciens strain has a BanH1 restriction enzyme as a bacterium belonging to the genus Bacillus. In addition, barnase, which is a natural antibiotic, and barstar and plantazolicin, antibiotics that have selective activity against anthrax have been actively studied (Molohon KJ, Melby JO, Lee J, Evans BS, Dunbar KL, Bumpus SB , Kelleher NL, Mitchell (2011). "Determination and Interception of Biosynthetic Intermediates for the Plantazolicin Class of Highly Discriminating Antibiotics" ACS Chem. Biol. 6 (12): 1307-1313, doi: 10.1021 / cb200339d). (1943), "Fukumoto (1943)," Isolation of bacterial secreting potent amylases and their distribution. "Journal of the Agricultural Chemical Society of Japan (in Japanese) 19: 487503.). The elongated endospore forms on paracentral or subterminal, and has non-swollen sporangia. It is also characterized by a peritrichous flagella motility and a thin film on its surface when growing in a liquid medium. It is well known that NaCl grows up to 12% and grows well at a temperature of 15 ~ 45 ℃ (Ruiz-Garcia, C., Bejar, V., Martinez-Checa, F., Llamas, I. & Quesada, E. 2005). Bacillus velezensis sp. Nov., Asurfactant- producing bacterium isolated from the river Velezin Malaga, southern Spain. Int. J. Syst. Evol. Microbiol., 55, 191-195. Since 2012, Bacillus velezensis has been recognized as the same organism as B. amyloliquefaciens and is now used as B. amyloliquefaciens in the academia (Li-Ting Wang, Fwu-Ling Lee, Chun-Ju Tai and Hsiao-Ping Kuo , 2008, Bacillus velezensis is a heterotypic synonym of Bacillus amyloliquefaciens. International Journal of Systematic and Evolutionary Microbiology (2008), 58: 671675., Leibniz Institute, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, 2012, Bacterial Nomenclalture UP-TO -DATE (APPROVED LISTS, VALIDATION LISTS). Pp. 17.). Many Bacillus strains are Plant growth-promoting rhizobacteria included in (PGPR) format (Pamela Calvo to the plant ^{rhizosphere, Ernesto OrmenEsperanza MartiDoris Zuniga *, 2010} , Characterization of Bacillus Isolates of Potato Rhizosphere from Andean Soils of Peru and Their Potential PGPR Characteristics. Brazilian Journal of Microbiology 41: 899-906). It was found that B. amyloliquefaciens produced potatoes and tomatoes with excellent crop growth, while it inhibited pathogens and improved soil environment by producing volatile organic compounds. (AureBailly and Laure Weisskopf *, 2012, The modulating effect of bacterial volatiles on plant growth. Plant Signaling & Behavior 7: 1, 17; January 2012; G 2012 Landes Bioscience., Kun Hao, Pengfei He, Jochen Blom , Christian Rueckert, Zichao Mao, Yixin Wu, Yueqiu He, and Rainer Borriss, 2012, The Genome of Plant Growth-Promoting Bacillus amyloliquefaciens subsp. plantarum Strain YAU B9601-Y2 Contains a Gene Cluster for Mersacidin Synthesis. Omar A. Almaghrabi *, Samia I. Massoud, Tamer S. Abdelmoneim, 2013, Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions. Saudi Journal of Biological Sciences (2013) 20, 5761).

  On the other hand, the temperature of the lethal phase of the apple was different from that of the development stage of the apple, and the temperature of 10% lethal temperature was 2.2 ℃ and the lethal temperature was 4.1 ℃. (Cited again, O Sung Do, 2004, Fruit-water temperature physiology, p. 153-162). It is difficult to bloom due to low temperature and frost in the spring, and it is difficult to form pollen because the number of petals is not changed or the formation of cancer surgery is not done properly, and normal correction is not made. In 2013, a farmhouse in Cheongsong-gun reported that the frost rate was lowered by more than 80% compared to the previous year (Jeongseo, 2004. 06. 03, The temperature dropped to below zero in May for two months, and apples, pears, and other fruit trees were seriously cold-weathered, and 1,882 farms totaled 1,709.1ha and 10.5ha damaged. 06. 09, Cheongsong Appeal to support the cold weather, Korean farmers' newspaper). With the sudden low temperature phenomenon in spring in 2013, Gyeongbuk province announced on July 9, 2013 that the area of the fruit field in the province that suffered from cold weather in the first half reached 2,075.. The damage areas of fruit species were 687.1 자 for plum, 478.4 포도 for grape, 339.7 배 for pear, 326.9 복 for peach, 201.3 사과 for apple, 26.2 살 for apricot, 4.8 매 for plum and 2.5 감 for plum. Yeocheon was the most affected area by 593., Followed by 318.4 의, 316.4 상, 316.1 김, and 142.8 성.

There was no cold weather in 2012, the damage area in 2011 was 9,573 ㏊, and in 2010 it was 3,477 ㏊. Gyeongbuk province decided to send the restoration support expenses of 3,450 million won including the government expense and the local expenses to the area where the cold-weather occurred (Seon Dae-sung, 2013 07. 09, Researcher disaster news). In this way, the damage of cold weather is caused by large damage of large area almost every year.

  Therefore, development of an environmentally friendly microorganism preparation for prevention and control of apple cold is considered to be an urgent task.

Korean Patent No. 10-0805044

Accordingly, the inventors of the present invention have conducted research and development of a method for preventing or controlling apple cold by biological methods using domestic indigenous microorganisms, and have succeeded in isolating B. amyloliquefaciens OKG-3 strain, And confirmed the possibility of development as a microorganism preparation for prevention or control of apple cold by using this strain, thereby completing the present invention.

  Accordingly, it is an object of the present invention to provide a novel strain of Bacillus amyloliquefaciens having an excellent prevention or control activity against cold damage to crops such as apples, pears and the like.

  The present invention also provides a microorganism preparation, a microbial pesticide or a microbial fertilizer containing the strain.

In order to achieve the above object, the present invention is a B. amyloliquefaciens KACC 92117P strain having a preventive or controlling activity against apple cold.

  Another embodiment of the present invention is a microorganism preparation, a microbial pesticide, or a microbial fertilizer containing the above-mentioned strain, and more preferably, for prevention or control of apple cold damage.

  The details of other embodiments are included in the detailed description and drawings.

According to the present invention described above, it is possible to provide a novel Bacillus amyloliquefaciens strain having an excellent preventive or controlling activity against cold damage to crops such as apples, pears or strawberries. It is also possible to provide a microorganism preparation, microbial pesticide or microbial fertilizer containing the strain.

  Accordingly, the present invention can effectively prevent or control the occurrence of cold weather caused in crops such as apples, pears, strawberries, and the like.

Brief Description of the Drawings Fig. 1 is a diagram showing a colony morphology and a representative microorganism selection for strain identification in a solid medium for Bacillus amyloliquefaciens OKG-3 strain according to the present invention (temporarily denominated Strain OK178-B1 for convenience) It's a picture,
FIG. 2 is a schematic diagram showing an example of the partial 16S rRNA gene sequence (1,393 bp) analysis result of the Bacillus amyloliquefaciens OKG-3 strain according to the present invention,
FIG. 3 is a graph showing the similarity analysis (NCBI / RDP BLAST homology search [NCBI BLAST Search: http: //blast.ncbi.nlm.) By the 16S rRNA sequence of Bacillus amyloliquefaciens OKG-3 strain according to the present invention . nih.gov/Blast.cgi ], [RDP BLAST Search: http://rdp.cme.msu.edu/seqmatch/seqmatch_intro .jsp]),
4 is a schematic diagram showing an example of the partial gyr B gene transfer sequence (1,035p) analysis result of the Bacillus amyloliquefaciens OKG-3 strain according to the present invention,
5 is a phylogenetic tree diagram showing an example of phylogenetic location of Bacillus amyloliquefaciens OKG-3 strain according to the present invention,
FIG. 6 is a schematic diagram showing the homology of B. amyloliquefaciens (DQ309294) and OKG-3, which are located in the same branch in FIG. 5,
FIG. 7 is a photograph showing an example of the growth of apple cultivated according to the control of the damage-free packaging of the microorganism preparation containing Bacillus amyloliquefaciens OKG-3 strain according to the present invention, ,
FIG. 8 is a photograph showing an example of the symptom of apple cold-weather in a control, an untreated control, and a treatment area of a microorganism preparation containing Bacillus amyloliquefaciens OKG-3 strain according to the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail.

The present invention is a Bacillus amyloliquefaciens KACC 92117P strain having a preventive or controlling activity against apple cold.

That is, the present invention relates to a strain having a preventive or controlling activity against cold damage occurring in a crop such as apple, pear or strawberry. Here, the cold weathering may be an apple, and the present invention may be a Bacillus amyloliquefaciens KACC 92117P strain for prevention or control of apple cold damage.

The inventors of the present invention have been studying a microorganism preparation capable of preventing or controlling apple cold by using native indigenous microorganisms, and found that B. amyloliquefaciens OKG-3 strain , Which was named as Bacillus amyloliquefaciens OKG-3 strain. This strain was deposited with the National Institute of Agricultural Science and Technology, National Institute of Agricultural Science and Technology on Jan. 08, 2016 and granted accession number KACC 92117P. Accordingly, the present invention is preferably a Bacillus amyloliquefaciens OKG-3 strain deposited under accession number KACC 92117P.

 The strain according to the present invention was isolated from the soil of the reclaimed soil in Ooxe-myeon, Gunsan-gun, Jeonbuk, among many soil samples collected from all over the country, and the strain having the same or similar characteristics as the strain according to the present invention could be obtained in other soil .

Further, another embodiment of the present invention can be a microorganism preparation, a microbial pesticide, or a microbial fertilizer containing the strain.

 As described above, the strain according to the present invention has an activity to prevent or control cold-weather, and it can be utilized for preventing or controlling cold-weather. Particularly, it is suitable to be used for preventing or controlling apple cold. The mode of use is not particularly limited and may be any form of microbial preparation, microbial pesticide or microbial fertilizer.

The present invention may be better understood by the following examples, which are for the purpose of illustrating the invention and are not intended to limit the scope of protection defined by the appended claims.

Example  One: Cold apple  Detection and isolation of microorganisms for prevention or control

First, we selected strains with high preventive or control activity against apple cold.

The apples used in the experiment were apple trees that were cold - weathered in apple cultivation farms in Nam - Sang - myeon, Geochang - gun, Gyeongsangnam - do. In order to isolate the bacterium that has the prevention or control activity against apple cold, dozens of soil samples were collected from all over the country, and apple trees suffered from apple cold damage were separately surveyed and prepared for public test. Subsequently, 1 g of each of the collected soil samples was suspended in sterilized distilled water, inoculated onto a Petri dish of a solid nutrient medium prepared in advance by a flat plate dilution method, and cultured in a thermostat at 30 ° C. for 2 to 3 days Pure separation.

FIG. 1 is a photograph showing a representative strain selection process for the colony shape and gene analysis in the solid medium for the Bacillus amyloliquefaciens OKG-3 strain according to the present invention. The OKG-3 strain shown here was obtained from Gunsan It is separated from the soil of the reclaimed land of Ooxeon-myeon.

Example  2: Identification of the strain

In order to identify OKG-3 strains isolated in Example 1, physiological and biochemical tests were performed (BERGEY's Manual of Systemic Bacteriology).

As a result, Nutrient Agar formed a coat with a yellowish milky colony (Creamy white) on the medium, and the morphological characteristic was aerobic bacterium. The minimum medium M9 (NaH ₂ HPO ₄ 7H ₂ O 12.8 g, KH ₂ PO ₄ 3 g, NaCl 0.5 g, NH ₄ Cl 1 g, 2 mM MgSO ₄ , 0.1 mM CaCl ₂ , 0.2% glucose, 0.5% casamino acid acid / H ₂ O 1) medium. Other physiological biochemical properties are summarized in Table 1 below.

 Physiological and biochemical characteristics of OKG-3 strain according to the present invention Survey item characteristic Cell type Bacillus Motility positivity Gram stain positivity Lactose positivity Glycogen ND Melibiose positivity Methyl alpha-D-glycoside positivity D-Raffinose positivity D-Turanose positivity Gelatin liquefaction positivity Tween 20 (Tween 20) positivity Tween 80 (Tween 80) ND Oxidase positivity Starch hydrolysis positivity Arginine dihydrolase < RTI ID = 0.0 > voice (ONPG, o- nitrophenyl- beta -D-galactopyranoside) voice

On the other hand, for the identification of more accurate strains, we performed phylogenetic classification of strains by 16S rRNA gene and gyr B gene sequence.

For this, OKG-3 strain was cultured in a nutrient broth for 72 hours with shaking, and DNA was extracted from the cells. PCR was performed using primers: 27F, 5'-AGAGTTTGATCCTGGCTCAG-3 'and 1492R, 5'- GGTTACCTTGTTACGACTT-3 '/ 30 cycles: 95 2 min, 94 30 sec, 58 30 sec, 72 45 min, 72 5 min, then 4 ℃ ∞), gyr B PCR (UP-1S (5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3') And amplified with UP-2R (5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3 ') / 30 cycles: 95 3 min, 95 30 sec, 61 30 sec, 72 1 min, 72 10 min, then 4 ° C) Qiagen), and the PCR products were analyzed by sequencing using DNA Sequencer 3730xl (Applied Biosystems). The nucleotide sequence was homologous in a database of NCBI / Genebank and Ribosomal Database Project II (RDP II) (Jukes, TH & Cantor, CR, 1969. Evolution of protein molecules. In Mammalian protein metabolism, 132 Edited by HN Munro, New York: Academic Press.).

As a result, the nucleotide sequence of the 16S rRNA sequence of the OKG-3 strain was as shown in Fig. 2, and the similarity analysis of the effective bacteria and the related bacteria by the 16S rRNA sequence was as shown in Fig. The OKG-3 strain was analyzed for the 16S rRNA sequence of 1,393 bp. NCBI / RDP BLAST homology was found to be 99.86% identical to the B. amyloliquefaciens standard strain and 16S rRNA sequence. In addition, Bacillus subtilis subsp. subtilis , Bacillus mojavensis , Bacillus vallismortis , Bacillus subtilis subsp. and spizizenii , and they were predicted as one of these strains.

Therefore, the same as the OKG-3 strain was subjected to re-identified by the gyr B gene, gyr B gene sequence is equal to [4], phylogenetic position [Figure 5]. The results of [Figure 5] show that Bacillus amyloliquefaciens (DQ309294) and the OKG-3 strain are as shown in Fig.

According to the phylogenetic tree showing the taxonomic position (philogenetic tree) analysis, as shown in Fig. 5] (Saitou, N. & Nei , M., 1987. The neighbor-joining method:. A new method for reconstructing phylogenetic trees Mol . Biol. Evol., 4, 406-425.), OKG-3 strain of the present invention to form a branch such as B. amyloliquefaciens strains, the B. amyloliquefaciens, B. atrophaeus, B. vallismortis type strain and The same group was formed. As a result of homology comparison with B. amyloliquefaciens strains located on the same branch, only four strains were found to be identical and more than 99% identical, and OKG-3 strains were judged to be B. amyloliquefaciens (Goodfellow, M. et al . , Manfio, GP & Chun, J., 1997. Towards a practical species concept for cultivable bacteria: In The Units of Biodiversity-Speciesin Practice , pp. 25-59 Edited by MF Claridge, HA Dawah & Hall, Stackebrandt, E. & Goebel, BM, 1994. A place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species in bacteriology. Int . J. Syst. Bacteriol., 44, 846-849. . Therefore, the OKG-3 strain according to the present invention was finally named as B. amyloliquefaciens OKG-3 strain.

Example  3: Preparation of microorganism culture medium and liquid type microorganism preparation

This example is for preparing a medium having the components and compositions most suitable for culturing the OKG-3 strain.

The medium for culturing or culturing the strain has a direct effect on the product cost of the microbial product. Particularly, since peptone and yeast extract as raw materials for medium are expensive to purchase raw materials, it is important to prepare a medium having components and compositions most suitable for culturing the strain.

Accordingly, in this experiment, Bacillus amyloliquefaciens OKG-3 strain was cultured in a minimal nutrient source to reduce the production cost. To this end, the present inventors prepared a medium prepared by mixing Glucose (Samchun Pure Chemical Co., Ltd.), Yeast extract (DSM Limited Company, Netherlands) and Peptone (Duchefa Biochemie, Netherlands) And each 100 ml of the Bacillus amyloliquefaciens OKG-3 strain previously cultured according to the present invention was inoculated therein, followed by incubation for 48 hours in a shaking incubator at 30 ° C.

Composition of medium for selecting medium for optimal microbial culture division Medium composition
(H ₂ O Per liter  Addition amount) division Medium composition
(H ₂ O Amount per liter ) division Medium composition
(H ₂ O Per liter  Addition amount) A Yeast extract 5.0 g
Glucose 3.0 g
Peptone 5.0 g A-1 Diluted to 1/2 of A A-2 Diluted to 1/4 of A B Yeast extract 5.0 g
Glucose 3.0 g B-1 Diluted to 1/2 of B B-2 Diluted to 1/4 of B C Glucose 3.0 g
Peptone 5.0 g C-1 Diluted to 1/2 of C C-2 Diluted to 1/4 of C D Yeast extract 5.0 g
Peptone 5.0 g D-1 Diluted to 1/2 of D D-2 Diluted to 1/4 of D E Yeast extract 5.0 g E-1 Diluted to 1/2 of E E-2 Diluted to 1/4 of E F Peptone 5.0 g F-1 Diluted to 1/2 of F F-2 Diluted to 1/4 of F G Yeast extract 1.0 g
Glucose 10.0 g
Peptone 2.0 g I Glucose 10.0 g
Peptone 2.0 g K Yeast extract 1.0 g H Yeast extract 1.0 g
Glucose 10.0 g J Yeast extract 1.0 g
Peptone 2.0 g L Peptone 2.0 g

The results are shown in Table 3 below. That is, the B and G compositions of the medium of Table 2 were the best grown overall [Table 3]. When the results of Table 3 below were combined, each strain was found to be the most important factor for growth of Yeast extract, followed by Glucose, and Peptone was found in B. amyloliquefaciens OKG-3 strain It does not seem to be a big help for growth.

Accordingly, it can be confirmed that the Bacillus amyloliquefaciens OKG-3 strain of the present invention is most economical and effective in culturing with a composition of 5.0 g of yeast extract and 1 liter of glucose 3.0 g / H ₂ O. The medium of this composition was named YG medium (Yeast Glucose media).

 Growth of Bacillus amyloliquefaciens OKG-3 strain by composition of each medium division Strain Degree of growth / Remarks division Strain Degree of growth / Remarks division Strain Degree of growth / Remarks A ++ A-1 ++ A-2 + B +++ B-1 ++ B-2 + C + C-1 - C-2 - D + D-1 + D-2 - E + E-1 + E-2 + F - F-1 - F-2 - G +++ I - K + H ++ J + L -

   -; Strain growth can not be confirmed with eyes, +; Slight increase, ++; Medium, +++; It grows very well

Thus, the culture of the strain according to the present invention was based on the YG liquid medium. The colony formed in the YG solid medium was inoculated into 100 ml of YG liquid medium and cultured for 48 hours. For this cultivation, the seed culture obtained above was inoculated and cultured in a 3 L Erlenmeyer flask containing 1.5 L of YG liquid medium. Culture conditions were shake cultured at 30 to 150 rpm for 48 hours. The culture was centrifuged to remove the culture medium, and the cells were recovered. The recovered cells were suspended in sterile distilled water so that the number of microorganisms was 10 ⁹ to 10 ¹⁰ cfu / ml, stored at 4 ° C, and used as a microbial preparation solution. The microbial preparation was diluted to 500 ml with 10 ⁶ to 10 ⁸ cfu / ml of the stock solution, and the microorganism preparation was prepared by mixing nutrients, trace elements, and other additives.

Example  4: Activity test of manufactured microorganism preparation

The microorganism preparation obtained from Example 3 was cultivated at 660 m ² (200 pyeong) among 3,960 m ² (1,200 pye) of apple orchard soil located in Nam Sang-myeon, Geochang-gun, Gyeongsangnam-do. In late April and late May of 2013, the cold temperatures of the flowers continued for more than five days, resulting in short apple blossoms and severe damage to the flowers. Then, the microbial agent was diluted 1,000 times and treated first with 1 bottle per 660 m ² (200 pyeong) of the first cold weather prevention or control agent on May 10, 2013, And the results were observed. Table 4 shows the results of the analysis of the effect of preventing or controlling the cold damage caused by the processing of microorganism-treated apple, untreated apple and microorganism.

Investigation of damage caused by apple micro-organisms division Flower picking  Immediately after closure () harvest Seedling number (dog) Fall rate
(%) Playing rate
(%) Post-harvest weight (g) Hyperbolic (Cm) Sugar content
( brix ) horizontal Vertical Cold weather
With and without 172 160 6.98 93.02 409.1 10.2 8.5 13.3 171 158 7.60 92.40 413.8 9.8 8.7 13.4 173 162 6.36 93.64 413.7 10.2 9.4 14.2 Average 172.00 160.00 6.98 93.02 412.20 10.07 8.87 13.63 Increase rate % ) 100 100 100 100 100 100 100 100 Cold weather, no treatment 183 140 23.50 76.50 406.7 10.1 9.0 12.4 185 136 26.49 73.51 338.3 9.8 8.0 14.1 182 132 27.47 72.53 368.7 9.6 8.7 12.4 Average 183.33 136.00 25.82 74.18 371.23 9.83 8.57 12.97 Increase rate % ) 106.59 85 369.91 79.75 90.06 97.68 96.61 95.11 Cold weather, product processing 184 167  9.24 90.76 415.6 9.9 9.0 14.2 185 160 13.51 86.49 431.8 10.2 8.8 14.0 183 167  8.74 91.26 435.9 10.3 9.3 17.6 Average 184.00 164.67 10.50 89.50 427.77 10.13 9.03 15.27 Increase rate % ) 106.98 102.92 150.38 96.22 103.78 100.66 101.88 111.98

FIG. 7 is a photograph showing an example of the growth of apples cultivated by a microbial preparation containing Bacillus amyloliquefaciens OKG-3 strain according to the present invention and an untreated control. FIG. 3 is a photograph showing an example of the symptom of the apple cold-weather caused by the treatment and the untreated control (control) of the microorganism preparation containing the Bacillus amyloliquefaciens OKG-3 strain according to the invention.

In other words, as shown in FIG. 7, the treatment rate of the microorganism product of apples suffering from cold damage was higher than that of untreated fruits, and the fruit weight, the weight, the size and the sugar content after harvest were higher than those of the untreated fruits. [Table 4, Fig. 7].

While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, It will be apparent to those skilled in the art that changes may be made.

<110> okeong agricultural corporation <120> Bacillus amyloliquefaciens strain and microbial agent for the          prevention and control in accordance with this cold-weather          damage <130> P15094 <140> 10-2016-0009078 <141> 2016-01-26 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 1393 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > KACC 92117P (Bacillus amyloliquefaciens OKG-3) <400> 1 gtcgagcgga cagatgggag cttgctccct gatgttagcg gcggacgggt gagtaacacg 60 tgggtaacct gcctgtaaga ctgggataac tccgggaaac cggggctaat accggatggt 120 tgtctgaacc gcatggttca gacataaaag gtggcttcgg ctaccactta cagatggacc 180 cgcggcgcat tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac 240 ctgagagggt gatcggccac actgggactg agacacggcc cagactccta cgggaggcag 300 cagtagggaa tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga 360 aggttttcgg atcgtaaagc tctgttgtta gggaagaaca agtgccgttc aaatagggcg 420 gcaccttgac ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa 480 tacgtaggtg gcaagcgttg tccggaatta ttgggcgtaa agggctcgca ggcggtttct 540 taagtctgat gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt 600 gagtgcagaa gaggagagtg gaattccacg tgtagcggtg aaatgcgtag agatgtggag 660 gaacaccagt ggcgaaggcg actctctggt ctgtaactga cgctgaggag cgaaagcgtg 720 gggagcgaac aggattagat accctggtag tccacgccgt aaacgatgag tgctaagtgt 780 tagggggttt ccgcccctta gtgctgcagc taacgcatta agcactccgc ctggggagta 840 cggtcgcaag actgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt 900 ggtttaattc gaagacacgc gaagaacctt accaggtctt gacatcctct gacaatccta 960 gagataggac gtccccttcg ggggagagt gacaggtggt gcatggttgt cgtcagctcg 1020 tgtcgtgaga tgttgggtta agtcccgcaa cgagcgcaac ccttgatctt agttgccagc 1080 attcagttgg gcactctaag gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg 1140 tcaaatcatc atgcccctta tgacctgggc tacacacgtg ctacaatgga cagaacaaag 1200 ggcagcgaaa ccgcgaggtt aagccaatcc cacaaatctg ttctcagttc ggatcgcagt 1260 ctgcaactcg actgcgtgaa gctggaatcg ctagtaatcg cggatcagca tgccgcggtg 1320 aatacgttcc cgggccttgt acacaccgcc cgtcacacca cgagagtttg taacacccga 1380 agtcggtgag gta 1393 <210> 2 <211> 1035 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > KACC 92117P (Bacillus amyloliquefaciens OKG-3) <400> 2 tggaattgac cattcatcgt gccggtaaaa ttcacgaaca agaataccgt catggcgatt 60 cacagtatcc attaaaagtg gtgggcgata ccgatagaac cggtactcgt gtccgtttct 120 ggccaagtgc cgagactttt agtcagacca tttttaatgt tgatattttg gcgcgtcgtt 180 tgcgtgaact gtcgttccta aacgcaggtg tacgtattgt gctgcgtgac gaacgcatta 240 atgccgagca tgtctttgat tatgaaggcg gtctgtctga gttcgttaaa tatattaacg 300 aaggcaaaac tcacctgaat gatattttcc attttactgc tgcgcaggct gataacggta 360 ttacggtaga agtcgcattg cagtggaatg attcttatca ggaaaatgtg cgttgcttta 420 ccaataacat cccgcaaaag gatgggggta cgcatttggc cggtttccgc gctgcgttga 480 cccgtggttt aaataactac atggacagcg aaaatatcct gaaaaaggaa aaagttgcgg 540 tatcgggtga tgatgcacgt gaaggtctga ctgccatcgt gtcagtcaaa gtacctgatc 600 caaaattctc ttcacagacc aaagaaaaac tggtgtcgag tgaagtgaaa accgctgttg 660 agcaagccat gaacaaggca ttttctgaat atttattgga aaatccacaa gcagccaagg 720 cgattgccgg caagattatt gatgcagcac gtgcgcgtga tgcagcgcgt aaagcccgtg 780 aaatgacacg tcgtaagagt gcgctagata ttgccggtct tccaggtaaa ctggccgatt 840 gtcaggaaaa agatccagct ttgtctgaac tgtacctggt cgagggtgat tctgcaggtg 900 gtcagccaa gcaaggtcgt aaccgtaaaa tgcaggcgat tttaccgctg aaaggtaaga 960 tcctgaacgt ggaacgtgcc cgttttgacc gcatgatttc ctctgctgaa gtcggtacgc 1020 tgattactgc actcg 1035

Claims (5)

Bacillus amyloliquefaciens OKG-3 KACC 92117P strain was used,
Wherein the strain comprises:
Which is characterized in that it is cultured in a medium consisting of 5.0 g yeast extract per liter of water (H 2 O) and 3.0 g of glucose (glucose), in order to prevent and control the cold damage of apple crops Microbial formulation.
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