KR20160041084A - Paenibacillus elgii 34-6 with antifungal activity to plant disease - Google Patents

Abstract

The present invention relates to a Paenibacillus elgii 34-6 strain with pest control activity against plant diseases and, more specifically, to a medium composition comprising a carbon source and a nitrogen source for culturing the Paenibacillus elgii 34-6 strain, to a composition comprising the strain and a culture product, and to a pest control method against plant diseases, using the same. Through the present invention, a novel Paenibacillus elgii 34-6 strain having excellent antifungal activity against plant diseases of various types is separated, a composition for controlling the plant diseases can be secured by obtaining the medium composition for the optimal growth of the strain and mass-production of bioactive materials, thereby the production of eco-friendly organic crops is increased, and marketability can be increased.

Description

Phenybacillus elgii 34-6 with antifungal activity to plant disease < RTI ID = 0.0 >

The present invention relates to a strain of Phenibacillus elgii 34-6 having plant disease control activity and more particularly to a culture medium containing a carbon source and a nitrogen source for culturing Fanny Bacillus strain BC34-6 , A composition comprising the strain and a culture product, and a method for controlling a plant disease using the same.

The land of our country is composed of more than 30% of the loess, so it is easy to get loess everywhere. Horticulture is an environmental condition in which the content of organic matter is insufficient compared with general soil, and the number of microorganisms is also about 100 times smaller at 2 × 10 ⁸ cfu / g. In the case of microorganisms living in loess, it may be considered that they have different metabolic characteristics from microorganisms inhabiting common soils in order to survive in a harsh environment.

When a microorganism is cultured for the purpose of mass production and commercialization of a useful microorganism preparation, the physiological activity inherent to the microorganism may vary depending on the medium composition and culture conditions. In particular, the high concentration There may be difficulties in production. Therefore, it is important to select an optimum medium that can replace the conventional high-price commercialization medium used for culturing microorganisms and maximize the production of physiologically active substances.

In the present invention, the medium composition and culture conditions for optimal growth of the novel strain showing the antifungal activity isolated from loess and the maximum production of the physiologically active substance, the above-mentioned physiologically active substance, the composition containing the same and the plant disease control method using the composition Respectively.

The present invention relates to a strain of Paenibacillus elgii 34-6 (Accession No. KACC 91971P).

Through the present invention, it was possible to identify a new strain from a yellow soil sample by analyzing the similarity of molecular base sequence, and named this as Fanny Bacillus elgii BC 34-6 ( Paenibacillus elgii 34-6) (KACC) on August 06, 2014.

The present invention also relates to a composition for controlling plant diseases, comprising the culture solution, the culture supernatant, the culture supernatant extract or the mixture thereof as an active ingredient.

In the present invention, the strain may be cultured by using any one or two or more selected from the group consisting of glucose, glycerin, sucrose, lactose, corn starch, water-soluble starch and chitin as a carbon source, You can use sucrose. It was confirmed that the growth of the strain and the antifungal activity were both highest when sucrose was used as a carbon source from one embodiment of the present invention.

In the present invention, the strain is not limited but any one or two or more selected from the group consisting of yeast extract, peptone, soybean meal, tryptone, and ammonium sulfate may be used as a nitrogen source. Preferably, soybean meal or yeast extract Can be used. From the present invention, it can be seen that when the sucrose is used as the carbon source and the soybean powder is used as the nitrogen source, the growth of the strain is the best and the highest antifungal activity is obtained when the yeast extract is used together with the nitrogen source there was.

In the present invention, the plant disease can be any one or two or more selected from the group consisting of red pepper pluck, red pepper anthracnose, cucumber gray mold, tomato gray mold, wilt disease and spotty disease.

In the present invention, the plant diseases include, but are not limited to, Fusarium oxysporum , Sclerotinia sclerotiorum , Colletotrichum gloeosporioides , pie And may be caused by one or more selected from the group consisting of Phytophthora capsici , Botrytis cinerea and Alternaria alternata .

In the present invention, the above extract is not limited, but can be obtained by extracting the culture supernatant with chloroform.

The present invention also relates to a method of controlling plant diseases, comprising treating the plant with the plant disease.

In the present invention, the plant disease can be any one or two or more selected from the group consisting of red pepper pluck, red pepper anthracnose, cucumber gray mold, tomato gray mold, wilt disease and spotty disease.

In the present invention, the plant diseases include, but are not limited to, Fusarium oxysporum , Sclerotinia sclerotiorum , Colletotrichum gloeosporioides , pie And may be caused by one or more selected from the group consisting of Phytophthora capsici , Botrytis cinerea and Alternaria alternata .

The present invention also relates to an eco-friendly agricultural material for controlling plant diseases comprising the above composition. In the present invention, the agricultural material is useful for growth of a plant, and may be used including, but not limited to, a component which does not have harmful effects on the plant.

Through the present invention, the Fanny Bacillus strain BC34-6 strain having excellent antifungal activity against various kinds of plant diseases was newly isolated, and by establishing the optimum growth of the strain and the medium composition for mass production of physiologically active substances, It is possible to secure a composition for the environment, thereby increasing the production amount of environmentally friendly organic crops and increasing the commerciality.

Figure 1 shows the phylogenetic tree of Fanny Bacillus strain BC34-6 strain,
Fig. 2 shows the result of measurement of antifungal activity against a plant pathogenic bacterium of Fanny Bacillus strain BC34-6 culture supernatant. The top of the petri dish is the culture supernatant.
Fig. 3 shows the result of measurement of antifungal activity against the plant pathogenic bacteria according to the experimental group of Table 4 of the Fanny Bacillus strain BC34-6 culture supernatant.
Fig. 4 shows the results of analysis of the molecular weight of Fanny Bacillus ELISA BC34-6 antifungal bioactive substance by a liquid chromatography mass spectrometer.
Fig. 5 shows the results of the comparison of the control effect against the anthrax anemia of the pepper Bacillus eligris BC34-6 medium.

Hereinafter, the present invention will be described in detail with reference to examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited by the following examples.

[Example 1] Isolation and identification of microorganisms derived from loess for controlling plant diseases

Gwangju metropolitan city and Jeollanamdo area have total 165 grains. A number of useful microorganisms having phytopathogenic fungicidal activity were isolated from yellow soil samples and cultured in TSB (tryptic soy broth) medium (17 g / L Pancreatic Digest of casein, 3 g / L Papaic Digest of Soybean, 2.5 g / L dextrose, 5 g / L sodium chloride and 2.5 g / L potassium phosphate) and stored at -80 ° C.

Identification of microorganisms was carried out by a molecular phylogenetic method through comparison of 16S rRNA nucleotide sequences. L of Pancreatic Digest of casein, 3 g / L of Papaic Digest of Soybean, 2.5 g / L of Dextrose, 5 g / L of sodium chloride, 2.5 g / L of potassium phosphate) at 37 ° C. in a tryptic soy broth medium Were collected and suspended in 100 μL of STES buffer (0.4 M NaCl, 0.2 M Tris-HCl (pH 7.6), 0.01 M EDTA, 1% SDS). A glass bead was added to the suspended sample, followed by disruption with a micro-tube mixer (MT-360) for 5 minutes to elute the cytoplasm. The cell extract was suspended in 200 μL of pH 8.0 TE buffer (Tris-EDTA buffer) and 200 μL of phenol / chloroform, and centrifuged at 12,000 rpm for 5 minutes. After 5 μl of RNase A (Promega, USA) was added to the supernatant, the mixture was reacted at 37 ° C for 1 hour, added with chloroform, and then centrifuged at 12,000 rpm for 5 minutes. The supernatant of the centrifuged sample was washed twice with 70% ethanol and then vacuum-dried with SpeedVac (Hanil, Korea). The final recovered DNA was stored in sterile distilled water.

100 ng of the recovered DNA sample, 50 μL of the 0.1 mM 27F (5'GTTTGATCCTGGCTCAG 3 ') primer, 50 μL of the 5'TACCTTGTTACGACTTCA 3' primer, 50 μL of 1.5 mM magnesium chloride (Promega, USA), 10 mM dNTPs (Promega, USA) and 2 U Taq DNA Amplification Enzyme (Promega, USA), and amplified 30 times at 94 ° C for 5 minutes to obtain 16S rRNA.

The 16S rRNA base sequence analysis was commissioned by Solgent Co. (Daejeon, Korea). As a result, the nucleotide sequence of the 16S rRNA of the newly isolated strain is shown in SEQ ID NO: 1, and the phylogenetic tree showing the molecular systematic taxonomic position is shown in FIG.

A new strain could be identified from the loess sample through analysis of the similarity of the biological sequence of the molecular system. Named as Fani Bacillus elgii BC-34 ( Paenibacillus elgii 34-6) And deposited on August 06, 2014.

[Example 2] Measurement of antifungal activity of Fanny Bacillus strain BC34-6 strain

The antifungal activity against plant pathogenic bacteria was examined using the supernatant of the culture obtained by cultivating the Fanny Bacillus ELISA BC34-6 strain isolated in Example 1 in a TSB (tryptic soy broth) medium at 30 ° C. 10 mL of the supernatant was administered to each of the plant pathogenic bacteria of Table 1, and the growth inhibitory effect of the causative bacteria was observed after 24 hours.

[Table 1]

As can be seen from FIG. 2, the strain of the present invention has a growth inhibitory effect on the causative bacteria of plant diseases, and particularly, it has been confirmed that the effect is very effective in controlling scleroderma (B) and anthracnose (C).

[Example 3] Medium composition for culture of Fanny Bacillus Elgius BC34-6 strain

Carbon source for culturing strains

1 g / L of sodium phosphate, 3 g / L of potassium phosphate dibasic, 0.5 g / L of sodium chloride, 1 g / L of ammonium chloride, 1 mL / L of 0.1 M calcium carbonate, 1 mL / L of 1 M magnesium sulfate Sigma) was added with 0.5 wt% yeast extract (Gibco), 10 g / L of each carbon source shown in Table 2 was added, and cultured at 30 DEG C and 150 rpm for 24 hours with stirring. For each experimental group, the degree of growth of the strain was measured at 600 nm by measuring the absorbance (OD ₆₀₀ ), and the degree of inhibition of mycelial growth was measured in the causative bacteria of Table 1 causing wilt, scleroderma, and red pepper anthracnose, The results are shown together in Table 2.

[Table 2]

¹ Antifungal activity evaluation scale: -: No inhibition, #: 0.2 mm inhibition, ##: 0.4 mm inhibition, ###: Inhibition by 0.5 mm or more.

As shown in Table 2, when the sucrose or chitin was used as the carbon source, the strain best survived and the antifungal activity was highest when sucrose and glycerol were used. From these results, it was confirmed that when sucrose is used as a carbon source, the strain grows best and exhibits a high antifungal activity.

A nitrogen source for culturing the strain

Based on the carbon source results, a carbon source was fixed with 10 g / L sucrose and added to the basal medium used in the carbon source experiment. 5 g / L of each nitrogen source shown in Table 3 was added and cultured at 30 DEG C and 150 rpm for 24 hours with stirring. For each experimental group, the degree of growth of the strain was measured at 600 nm by measuring the absorbance (OD ₆₀₀ ), and the degree of inhibition of mycelial growth was measured in the causative bacteria of Table 1 causing wilt, scleroderma, and red pepper anthracnose, The results are shown together in Table 3.

[Table 3]

¹ Antifungal activity evaluation scale: -: No inhibition, #: 0.2 mm inhibition, ##: 0.4 mm inhibition, ###: Inhibition by 0.5 mm or more.

As shown in Table 3, when the soybean powder was used as the nitrogen source, the strain was the best grown and the antifungal activity was the highest when the yeast extract was used.

[Example 4] Antifungal bioactive substance derived from Fanny Bacillus strain BC34-6 strain

10 g / L sucrose, 5 g / L yeast extract (Gibco), 6 g / L sodium diphosphate, 3 g / L potassium diphosphate, 0.5 g / L sodium chloride, 1 g / L, 0.1 M calcium carbonate 1 mL / L, and 1 M magnesium sulfate 1 mL / L (Sigma), and then cultured at 30 DEG C and 120 rpm for 60 hours inoculated with Fanny Bacillus strain BC34-6. The culture supernatant obtained through the above cultivation was treated as shown in Table 4, and the degree of inhibition of mycelial growth was measured in the causative bacteria of Table 1 causing wilt disease, sclerotinia and pepper anthracnose, .

[Table 4]

¹ Antifungal activity evaluation scale: -: No inhibition, #: 0.2 mm inhibition, ##: 0.4 mm inhibition, ###: Inhibition by 0.5 mm or more.

As can be seen in Table 4, the antifungal activity was highest in the chloroform extract (c) or the protein solution dialysis (f) treated group after ammonium sulfate precipitation of the heating culture supernatant at 100 ° C for 15 minutes (FIG. 3).

(e) Although the enzyme lost its activity in the experimental group, the major antifungal substance could be expected to be a peptide component, not an enzyme protein component, since the antifungal activity is weak. The main MS values were 1073.9, 1088.0, and 1102.4 Da, respectively, as a result of analyzing the samples of the experimental group by the mass spectrometer (manufacturer, country) (Fig. 4). ≪ / RTI >

[Example 5] Effect of culture medium of Fanny Bacillus strain BC34-6 on control of pepper anthracnose

The culture was inoculated with the Fanny Bacillus strain BC34-6 under the same conditions and culture conditions as in Example 4, and the culture solution was recovered. The effect of controlling the plant disease of the sample diluted with 1 × 10 ⁸ cells / mL of the culture broth by 1 × 10 ⁸ cells / mL was compared with the list of environmentally friendly agricultural and livestock products that are sold on the market for the control of anthrax. Future) and Bio Strong (Hyunnong Co., Ltd.).

The three specimens and the strains were treated with foliar treatment in a red pepper seedling experimental group (Asian seedling, Korea) grown for about 5 weeks, and each of the pepper anthrax bacteria was inoculated to induce pathology. As a control group, red pepper seedlings inoculated with anthracnose anthracnose of pepper without using samples and strains were used. In the experimental group, the disease began to appear after 7 days. There was no significant difference in the severity of the lesions at the beginning, but after 7 days from the onset of the disease, there was a difference in each test group.

As can be seen from FIG. 5, infection rate was about 70% in the control group (A), and it was confirmed that the pathogenesis occurred. (B), (C), and (F) of the Fanny Bacillus strain ELISA BC36-4 were significantly lower than the control (A) It is confirmed that the performance is better in terms of control effect.

Depositor Name: National Institute of Agricultural Science

Accession number: KACC91971P

Funding date: 20140806

<110> Jeonnam Bioindustry Foundation <120> aenibacillus elgii 34-6 with antifungal activity to plant disease <130> P1407148090 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1416 <212> RNA <213> Paenibacillus elgii 34-6 <400> 1 atacatgcaa gtcgagcgga cccttcgggg ttagcggcgg acgggtgagt aacacgtagg 60 caacctgcct gtaagactgg gataactacc ggaaacggta gctaagaccg gataagtgat 120 tctctcgcat gagaggatca agaaacacgg ggcaacctgt ggcttacaga tgggcctgcg 180 gcgcattagc tagttggtgg ggtaacggct caccaaggcg acgatgcgta gccgacctga 240 ggggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 300 agggaatctt ccgcaatgga cgcaagtctg acggagcaac gccgcgtgag tgatgaaggt 360 tttcggatcg taaagctctg ttgccaggga agaacgtcgt ggagagtaac tgctctgcga 420 atgacggtac ctgagaagaa agccccggct aactacgtgc cagcagccgc ggtaatacgt 480 agggggcaag cgttgtccgg aattattggg cgtaaagcgc gcgcaggcgg ccgcttaagt 540 ctggtgttta agcccgaggc tcaacctcgg ttcgcactgg aaactgggtg gcttgagtgc 600 aggagaggaa agcggaattc cacgtgtagc ggtgaaatgc gtagagatgt ggaggaacac 660 cgtggcgaa ggcggctttc tggcctgtaa ctgacgctga ggcgcgaaag cgtggggagc 720 aaacaggatt agataccctg gtagtccacg ccgtaaacga tgagtgctag gtgttagggg 780 tttcgatacc cttggtgccg aagtaaacac aataagcact ccgcctgggg agtacgctcg 840 caagagtgaa actcaaagga attgacgggg acccgcacaa gcagtggagt atgtggttta 900 attcgaagca acgcaagaac cttaccaggt cttgacatcc ctctgaatat cctagagata 960 gggtaggcct tcggggacag aggagacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1020 gagatgttgg gttaagtccc gcaacgagcg caacccttga acttagttgc cagcattgag 1080 ttgggcactc taagttgact gccggtgaca aaccggagga aggtggggat gacgtcaaat 1140 catcatgccc cttatgacct gggctacaca cgtactacaa tggccggtac aacgggaagc 1200 gaagtcgcga gatggagcca atcctaagaa agccggtctc agttcggatt gcaggctgca 1260 actcgcctgc atgaagtcgg aattgctagt aatcgcggat cagcatgccg cggtgaatac 1320 gttcccgggt cttgtacaca ccgcccgtca caccacgaga gtttacaaca cccgaagtcg 1380 gtggggtaac cgcaaggagc cagccgccga aggtgg 1416

Claims (11)

Fanny Bacillus strain BC34-6 ( Paenibacillus elgii 34-6) strain (Accession No. KACC 91971P). A plant disease controlling composition comprising the culture of the strain of claim 1, a culture supernatant, an extract of the culture supernatant, or a mixture thereof as an active ingredient. 3. The method of claim 2,
Wherein the strain is cultured by using any one or two or more selected from the group consisting of glucose, glycerin, sucrose, lactose, corn starch, water-soluble starch, and chitin as a carbon source. 3. The method of claim 2,
Wherein the strain is cultured by using one or two or more selected from the group consisting of yeast extract, peptone, soybean meal, tryptone, and ammonium sulfate as a nitrogen source. 3. The method of claim 2,
Wherein the plant disease is any one or more selected from the group consisting of red pepper pox, red pepper anthracnose, cucumber gray mold, tomato gray mold, wilt disease and spotty disease. 3. The method of claim 2,
The plants may be selected from the group consisting of Fusarium oxysporum , Sclerotinia sclerotiorum , Colletotrichum gloeosporioides , Phytophthora capsici ), Botrytis cinerea , and Alternaria alternata . &Lt; Desc / Clms Page number 20 &gt; 3. The method of claim 2,
Wherein the extract is obtained by extracting the culture supernatant with chloroform. 8. A method for controlling plant diseases, comprising the step of treating a plant having a plant disease with the composition of any one of claims 2 to 7. 9. The method of claim 8,
Wherein the plant disease is any one or two or more selected from the group consisting of red pepper pox, red pepper anthracnose, cucumber gray mold, tomato gray mold, wilt disease and spot flaccid disease. 9. The method of claim 8,
The plants may be selected from the group consisting of Fusarium oxysporum , Sclerotinia sclerotiorum , Colletotrichum gloeosporioides , Phytophthora capsici ), Botrytis cinerea cinerea and Alternaria alternata ). &lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt; 8. An eco-friendly agricultural material for controlling plant diseases, comprising the composition of any one of claims 2 to 7.

Images