KR101759521B1 - Composition for preventing plant storage disease and a control method of plant storage disease using the same - Google Patents

Abstract

The present invention provides a novel strain of Bacillus tequilensis BOR1 deposited with Accession No. KCTC18379P.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing plant storage diseases, and a method for controlling plant storage diseases using the same.

The present invention relates to a composition for controlling a plant disease, and more particularly, to a composition for controlling a disease caused by a plant disease.

Currently, domestic horticultural crop production amounted to 11 trillion won, but loss of about 3 trillion won occurred in the process of distribution after harvest. Generally, when temperature and humidity are not controlled when storing grains, fruits and vegetables, the growth of pathogens causes a great deal of damage to crops after harvest. The use of chemically synthesized pesticides in controlling these plant diseases has been increasing in Rio ) Convention and the like, and it is demanded to develop harmless and environment friendly pest control method, so that development of control methods using low toxicity and environmentally friendly pesticides to prevent corruption of storage fruits is important. The control method using biological pesticides derived from natural materials has the advantage of solving the problems of microbial agents and lower development cost than chemical pesticides. Accordingly, a method of controlling natural disease such as jasmonic acid, an amino acid and a derivative thereof such as β-aminobutyric acid, or a physical disease control method such as washing with UV and hot water Is being studied actively.

Korean Patent Registration No. 1332360 discloses a composition for preventing fruit-bearing diseases, which comprises an extract of Tianyans and a microbial strain.

However, until now, the development of biological pesticides derived from natural products is limited and only a few.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a composition for controlling a plant storage disease using microorganisms and a method for controlling a plant storage disease using the same. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, a new strain of Bacillus tequilensis BOR1 deposited with Accession No. KCTC18379P is provided.

 According to another aspect of the present invention, there is provided a composition for controlling a plant veterinary disease comprising the strain.

  According to another aspect of the present invention, there is provided a method of controlling a plant storage disease comprising treating the plant with the strain or the composition.

According to one embodiment of the present invention as described above, the microorganisms isolated from the surface of the fruits and vegetables can be used to realize a storage bottle control effect with excellent effect. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a photograph showing the antibacterial activity of a BOR1 strain isolated according to an embodiment of the present invention. FIG.
FIG. 2 is a graph illustrating the effect of isolating the BOR1 strain according to an embodiment of the present invention to prevent apple storage disease. FIG.
FIG. 3 is a graph illustrating the effect of isolating BOR1 strain according to an embodiment of the present invention on the prevention of orange and pear storage diseases.
FIG. 4 is an analysis of phylogenetic results according to the analysis of the nucleotide sequence of the BOR1 strain isolated according to an embodiment of the present invention.

Definition of Terms:

As used herein, the term "plant storage disease" is a disease that occurs in agricultural products that are stored after harvesting. If the temperature and humidity are not controlled during storage, the propagation of pathogens is promoted, do. In general, diseases that occur during the distribution process of agricultural products, such as transportation diseases during transportation after harvest, market and retail stores, are called market diseases and diseases caused by fruits and vegetables among market diseases are fruits and vegetables.

As used herein, the term "gray mold rot" is a storage bottle caused by the plant pathogen Botrytis cinerea , which changes from leaf edge to brown and wavy wrinkles around the lesion.

As used herein, the term "green mold rot" is a storage disease caused by the plant pathogen Penicillium expansum . If the fruit skin is injured in the post-harvest storage or in the box in transit, It develops.

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, a new strain of Bacillus tequilensis BOR1 deposited with Accession No. KCTC18379P is provided.

The strain may have a nucleic acid sequence represented by SEQ. ID.

According to another aspect of the present invention, there is provided a composition for controlling a plant veterinary disease comprising the strain.

The composition for controlling a vegetative depot disease comprising the strain can be prepared as a microbial agent in liquid or granular form depending on the use of the culture. The formulation of microbial pesticides is well described in the prior art (Korean Patent No. 0314323). For example, in the case of a liquid preparation, Bacillus tequilensis of 10 ⁹ cfu / ml or more, 1 w / v of glycols, 1 w / v% of NaCl, 1 w / v% of Tween 80 and potassium sorbate, ( Bacillus tequilensis ) of 10 ⁹ cfu / ml or more, NaNO _{3 of} 0.01 wt%, glucose of 1.0 wt% and the remainder of the zeolite It is possible.

The plant storage bottle may be caused by Penicillium expansum , Botrytis cinerea , Fusarium solani , Pythium ultimum or Alternaria alternata , The plant may be an apple, a pear, a red pepper, a cabbage, a radish, a carrot, a garlic, an onion, a cucumber, a melon, a tomato or a citrus fruit, and the storage bottle may be a storage disease, a transportation disease or a market disease.

According to another aspect of the present invention, there is provided a method of controlling a plant storage disease comprising treating the plant with the strain or the composition.

The active ingredient of the biocidal pesticide containing the strain according to the present invention is preferably 0.001 to 90% of the total microorganism composition or diluted 100 to 500 times with the bacterial culture broth. In order to control the plant disease control agent as described above to control the gray mold fungus, root disease, spot fungus disease, pollen fungus, root rot fungus, black fungus disease and blue fungus disease, It is preferable to treat it by a foliar treatment method. The microbial agent may be used as a general pesticide such as a plant disease controlling agent, a seed coating agent, a microbial nutrient, and a soil improving agent.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Isolation of strain

According to one embodiment of the present invention, Bacillus strains showing antibacterial activity on the surface of fruit vegetables were separated.

Specifically, commercially available oranges were purchased and the orange peel was cut into small pieces in order to separate the Bacillus strain from the surface, and 50 ml of sterilized distilled water was added thereto and finely ground using a blender. 0.1 ml was taken from the sample, and 2 ml of 2X SG liquid medium (1.6% nutrient medium, 0.05% MgSO ₄ , 0.2% KCl, 1 mM CaNO ₃ , 0.1 mM MnCl ₂ , 1 M M FeSO ₄ and 0.1% glucose) Mixed and incubated at 37 [deg.] C for 3 days with shaking. ^10-1 times the 0.1 ml diluted culture liquid is plated on LB agar medium Colonies (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1% glucose, pH 7.0) and the resulting after incubation overnight at 37 ℃ incubator Respectively. A strain estimated to be Bacillus sp. Among the above-mentioned various colonies was selected, and plate culture was repeated to confirm that it had a unique shape by the naked eye. Microorganism observation confirmed the presence of a single strain, BOR1 was isolated.

Example 2: Identification of Strain

The BOR1 strain was identified using 16S rDNA sequence sequence analysis according to an embodiment of the present invention. The analysis was commissioned by the Korean Center for Microbiological Conservation (KCCM) and requested microbial identification services using the full nucleotide sequence analysis of the 16S rDNA gene. As a result of phylogenetic analysis according to the analysis of the nucleotide sequence of the strain, it was confirmed that the 16S rDNA sequence (SEQ ID NO: 1) of the strain showed 99% homology with Bacillus tequilensis , The strain BOR1 was identified as Bacillus tequilensis (Fig. 4). In addition, the isolated strain was deposited with KCTC, an international patent depository institution located in the Korean Institute of Bioscience and Biotechnology, 125, Yuseong-gu, Daejeon, on Apr. 23, 2015, and received the accession number KCTC18379P. The deposit shall be deposited under conditions satisfying all the requirements of the Budapest Treaty.

Experimental Example 1: Antimicrobial activity test for plant pathogenic bacteria

The antibacterial activity of BOR1 strain isolated according to one embodiment of the present invention was measured. Serenade ( Bacillus subtilis QST 713), a biopesticide of the US AgraQuest, has excellent plant disease control activity and is known as the most successful biocide. Therefore, the QST 713 strain isolated from the Serenade product and the BOR1 strain of the present invention Were compared. Plant pathogens used in the above-mentioned antibacterial activity tests were Botrytis cinerea KACC 40573 (gray mold fungus), Fusarium solani KCTC 6326 (root germ), Pythium ultimum KACC 40705 (root rot disease) and Alternaria alternata KCTC 6326 (black fungus) It was used by the Resource Center and the Korean Agricultural Microbiology Resource Center.

The isolated BOR1 strain and QST 713 strain were inoculated into TSB (Difco) liquid medium and cultured overnight at 37 ° C at 220 rpm. A filter paper disc (8 mm) was placed on one side of a PDA (Potato dextrose broth, Difco) agar medium, and 10 μl of the culture broth of each strain was inoculated thereon. Thereafter, the mycelial fragments obtained from the cultivated plant pathogenic bacterium grown in the PDA medium at a distance of about 3 cm were inoculated and cultured at 30 ° C. for 3 days or more to inhibit the growth by the strain when the plant pathogenic bacteria grow to the edge Was measured.

As a result, it was confirmed that the BOR1 strain of the present invention inhibits the growth of most plant pathogens similarly to the strain QST 713 (Fig. 1).

Experimental Example 2: Prevention of apple storage disease

The effect of preventing the storage of BOR1 strain isolated according to one embodiment of the present invention was measured.

In particular, most fruits are damaged by various diseases during storage after harvest. Among them, gray mold rot (green mold rot) and green mold rot (green mold rot) Causing a great deal of damage. Especially, apple can not use pesticides at all after harvesting, so we need a new pesticide substitution technology that can prevent fruit rot after harvesting, so we observed the effect of preventing the storage diseases in apples. In order to observe the inhibitory effect of the BOR1 strain having plant pathogenic growth inhibitory activity on the apple together with the pathogenic bacteria, firstly, the apple was washed cleanly in running water and dried. Then, a toothpick of 5 mm depth was drilled with a triangular hole (1 g / l molasses, 10 g / l molasses, 10 g / l molasses and 1 g / l CaCO ₃ ) and cultured at 37 ° C for 3 days for 3 days to form BOR1 and QST 713 ㅧ 10 ⁹ CFU / ml) were treated with 20 ㎕ each, and sterilized water was used as a control. As a positive control, the QST 713 strain was used. Then, the treated strain and the sterilized water were absorbed into the apple, and then the mycelium of Penicillium expansum KCTC6434 (blue fungus germ) and Botrytis cinerea KACC 40573 (gray mold fungus) which grew strongly in the PDA medium was sterilized with 0.1% tween 20 Was inoculated with 20 쨉 l of the obtained solution. The BOR1 strain and the apple inoculated with the plant pathogenic bacterium were stored in an incubator at 20 ° C and 80% humidity, and then the symptom observed in the apple was observed 5 days after inoculation of the pathogen. The apples used in the experiment were rubrum and aquatic cultivars. The degree of disease of the blue mold was measured, the size of the symptom was measured, the rate of decay was measured, and the size of the symptom of the gray mold was observed. The size of the symptom was measured using the index shown in Table 1 below.

level Size of symptom 0 No disease One Disease less than 5 mm from the pathogen inoculated area 2 Disease 6-10 mm from pathogen inoculation site 3 11-15 mm disease outbreak from pathogen inoculation site 4 Disease 16-20 mm from pathogen inoculation site 5 21 mm or more from diseased area

As a result, the BOR1 strain of the present invention showed similar or lower disease severity than the QST 713 strain used as a pesticide, and the BOR1 strain exhibited excellent control activity against gray mold and blue fungus disease in apple (Fig. 2).

Experimental Example 3: Preventive effect of tangerine and pear storage

In accordance with one embodiment of the present invention, it was determined whether the isolate BOR1 exhibited the effect of controlling the storage disease in fruits other than apples.

Specifically, in the same manner as in Example 3, the test method was cleanly washed in water flowing through mandarin oranges and dried, and then holes of 5 mm in depth were drilled in three places using a toothpick on the surface of the fruit, and BOR1 and QST 713 cultures 10 < ⁹ > CFU / ml) and sterilized water, respectively. Then, 20 ㎕ of the solution obtained Botrytis cinerea KACC40573 (gray mold fungus), which was strongly grown in the PDA medium, was inoculated and cultured in an incubator at 20 캜 and 80% humidity. After 5 days of inoculation, The formed symptoms were observed. Likewise, the degree of disease in the mandarin and pear was measured using the indices in Table 1 above.

As a result, it was observed that the BOR1 strain of the present invention exhibited excellent disease control activity also in mandarin and pear (FIG. 3).

Experimental Example 4: Confirmation of the fixation ability of the strain on the fruit surface

The microorganism preparation for controlling the storage disease is advantageous to fix on the surface of the host as well as against pathogens. Therefore, the fruit surface fixing ability of the BOR1 strain isolated according to one embodiment of the present invention was observed.

Specifically, strain BOR1 was cultured in P3 medium for about 3 days, and the apple was immersed in a Bacillus culture (1 × 10 ⁷ CFU / ml) for 1 hour. The treated samples were dried for 1 hour and then cultured in an incubator at 80 ° C and humidity at 20 ° C for one week. To determine the ability of the strain to be fixed on the surface of the apple, the apple peel was finely cut and finely ground with a blender. The samples were serially diluted to 10 ^-1 to 10 ^-3 , stained on LB agar medium, And QST 713 and Bacillus subtilis isolates were used as controls.

As a result, the BOR1 strain of the present invention showed about 10 times higher number of strains than the other Bacillus subtilis as well as QST 713, confirming that the BOR1 strain has a better ability to fix the surface of the apple than other Bacillus strains. The results of measuring the fixation ability of the BOR1 strain on the apple surface are shown in Table 2 below.

Apple weight (g) Number of strains Number of strains per gram of apple Control group 200.12 0 0 QST713 185.94 0.66 ㅧ 10 ⁴ 0.35 ㅧ 10 ² Bacillus subtilis 248.26 0.07 ㅧ 10 ⁴ 0.03 ㅧ 10 ² BOR1 229.80 7.6 ㅧ 10 ⁴ 3.31 ㅧ 10 ²

As a result, various microorganisms having antimicrobial activity have been reported so far. However, in the present invention, a new Bacillus strain having an antifungal activity on an orange surface was isolated from microorganisms inhabit on the surface of fruit and vegetables, Antibacterial activity, storage disease control activity and apple surface fixing ability are excellent, it can be useful for controlling the storage diseases of fruit and vegetables.

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, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. I will understand. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Microbiology Resource Center (Korea) KCTC18379P 20150423

<110> KOREA RESEARCH INSTITUTE OF BIOSCIENCE AND BIOTECHNOLOGY <120> Composition for preventing plant storage disease and a control          method of plant storage disease using the same <130> PD15-5221 <160> 1 <170> KoPatentin <210> 1 <211> 1431 <212> DNA <213> Artificial Sequence <220> <223> Bacillus tequilensis BOR1 <400> 1 ctatcatgca gtcgagcgga agatgggagc ttgctccctg atgttagcgg cggacgggtg 60 agtaacacgt gggtaacctg cctgtaagac tgggataact ccgggaaacc ggggctaata 120 ccggatggtt gtttgaaccg catggttcaa acataaaagg tggcttcggc taccacttac 180 agatggaccc gcggcgcatt agctagttgg tgaggtaacg gctcaccaag gcaacgatgc 240 gtagccgacc tgagagggtg atcggccaca ctgggactga gacacggccc agactcctac 300 gggaggcagc agtagggaat cttccgcaat ggacgaaagt ctgacggagc aacgccgcgt 360 gagtgatgaa ggttttcgga tcgtaaagct ctgttgttag ggaagaacaa gtaccgttcg 420 gt ccgcggtaat acgtaggtgg caagcgttgt ccggaattat tgggcgtaaa gggctcgcag 540 ggggtttctt aagtctgatg tgaaagcccc cggctcaacc ggggagggtc attggaaact 600 ggggaacttg agtgcagaag aggagagtgg aattccacgt gtagcggtga aatgcgtaga 660 gatgtggagg aacaccagtg gcgaaggcga ctctctggtc tgtaactgac gctgaggagc 720 gaaagcgtgg ggagcgaaca ggattagata ccctggtagt ccacgccgta aacgatgagt 780 gctaagtgtt agggggtttc cgccccttag tgctgcagct aacgcattaa gcactccgcc 840 tggggagtac ggtcgcaaga ctgaaactca aaggaattga cgggggcccg cacaagcggt 900 ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg acatcctctg 960 acaatcctag agataggacg tccccttcgg gggcagagtg acaggtggtg catggttgtc 1020 gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttgatctta 1080 gttgccagca ttcagttggg cactctaagg tgactgccgg tgacaaaccg gaggaaggtg 1140 gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200 agaacaaagg gcagcgaaac cgcgaggtta agccaatccc acaaatctgt tctcagttcg 1260 gatcgcagtc tgcaactcga ctgcgtgaag ctggaatcgc tagtaatcgc ggatcagcat 1320 gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380 aacacccgaa gtcggtgagg taacctttta ggagccagcc gcgcgaaggt g 1431

Claims (7)

New Bacillus tequilensis BOR1 strain deposited with accession number KCTC18379P and having excellent fruit and vegetable surface fixing ability. The method according to claim 1,
Wherein the 16S rDNA has the nucleic acid sequence as set forth in SEQ ID NO: &lt; RTI ID = 0.0 &gt; 1. &lt; / RTI &gt; A plant derived by the strain of claim 1, which is caused by Penicillium expansum , Botrytis cinerea , Fusarium solani , Pythium ultimum or Alternaria alternata . A composition for controlling a storage disease. delete The method of claim 3,
Wherein said plant is apple, pear, red pepper, cabbage, radish, carrot, garlic, onion, cucumber, melon, tomato or citrus. The method of claim 3,
Wherein the storage bottle is a storage pest, transportation pest, or market pest. ( Penicillium expansum ), Gray mold fungus ( Botrytis cinerea ), Fusarium spp. ( Fusarium spp. ), Which comprises a step of treating a plant according to claim 1 or a composition according to any one of claims 3, solani ), Pythium ultimum or Alternaria alternata ( Pseudomonas aeruginosa ).

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