KR101541213B1 - Composition containing extracts of Streptomyces for preventing Xanthomonas spp. - Google Patents

Abstract

The present invention relates to a composition for inhibiting a pathogenic fungus belonging to the genus Zanthomonas, comprising an extract obtained by extracting actinomycetes of the Sequence Number 1 with ethyl acetate.
Since the actinomycetes extract according to the present invention has inhibitory activity against blight of blight of white blotch or blight of blight of spot blight of leaf blight, this ingredient can be used as pesticide or as a fermented product pesticide containing this substance.

Description

Compositions containing extracts of Streptomyces for preventing Xanthomonas spp.

The present invention relates to a composition for inhibiting a pathogenic fungus belonging to the genus Zanthomonas, comprising an extract obtained by extracting actinomycetes of the Sequence Number 1 with ethyl acetate.

Bacterial blight of rice is dormant in the stalks, roots, or sick stumps of rice, which are known as host plants during the wintering period, After being moved to the rice field by irrigation water, it penetrates into water holes, air holes and cut roots of rice leaves and is infected with rice. It propagates in infected rice and is transmitted by the second order. ~ The disease that occurs in the middle of August causes the rice to finally dry down, resulting in a decrease in the yield, which is very damaging to farmers.

Bacterial spotting is caused by Xanthomonas campestris pv. Vesicatoria, which occurs in seedlings and main fields. It forms brown to dark brown spots, leaves fall, and many lesions form. Symptoms appear on leaves, petioles, stems, trees and fruits. The early leaves of the infection appear as small brownish brown or amorphous small spots and form a light yellow band around them. When the disease progresses, the central part of the lesion becomes white, the lesion becomes brown or dark brown, and several lesions are combined to form a large lesion. Deeply, the leaves become dry or fall off. Leaflets and fruit are rounded with brown spots. When developed, they grow slightly to dark brown irregular shape. The diseased fruit does not decay, but the value of the product drops. Small streaks of dark brown water-colored beds are formed on the stem, and the center of the lesion becomes convex later.

On the other hand, rice, pepper and cabbage are cultivated crops of 1, 2 and 3 in Korea. Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas campestris pv. Vesicatoria (Xcv) is a pathogenic bacterium of rice blight and leaf spot blight of pepper, respectively. These pathogens are causing serious damage to crop yields in most Asian countries.

Therefore, both academia and industry are studying in depth the practical prevention and treatment of disease. For example, all the genomes of Xoo and Xcv became clear.

Chemical insecticides are widely used to control plant pathogens in agriculture. However, the use of chemical pesticides in recent years has raised many issues, including the possibility of causing environmental toxicity and antibiotic-resistant microorganisms. Therefore, biocides made from microorganisms or secondary metabolites from plants are preferred. In addition, international codes and agreements favor biological pesticides.

Korean Patent No. 10-2011-0070588

Thus, it has been developed effective natural physiologically active substances that inhibit the pathogenic fungus of the genus Zanthomonas.

The present invention provides a composition for inhibiting a pathogenic fungus belonging to the genus Zanthomonas, which comprises an extract obtained by extracting actinomycetes of the Sequence Number 1 with ethyl acetate.

The present invention relates to a composition for inhibiting pathogens of Xanthomonas comprising an extract obtained by extracting actinomycetes of the Sequence Number 1 with ethyl acetate.

The present invention also relates to a method for controlling Xanthomonas oryzae pathovar oryzae or Xanthomonas campestris pathovar vesicatoria using the above composition.

Since the actinomycetes extract according to the present invention has inhibitory activity against blight of blight of white blotch or blight of blight of spot blight of leaf blight, this ingredient can be used as pesticide or as a fermented product pesticide containing this substance.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a bar pie chart showing the first antibacterial activity screening of actinomycetes extract against (A) Xoo and (B) Xcv.
Figure 2 is the first screened antibacterial extract against (A) Xoo and (B) Xcv. The bars show the relative growth of (A) Xoo and (B) Xcv with each screening extract versus the growth of bacteria not treated with the extract.
Figure 3 relates to growth curves of (A) Xoo and (B) Xcv with antimicrobial extracts examined in a second growth inhibition screening.
Figure 4 is the growth curve of Xoo treated with 1172E10 for the measurement of GI50.
Figure 5 is a growth curve of Xcv treated with 1172E10 for the measurement of GI50.
6 relates to SEQ ID NO: 1.

The present invention relates to a composition for inhibiting pathogens of Xanthomonas comprising an extract obtained by extracting actinomycetes of the Sequence Number 1 with ethyl acetate.

The actinomycetes extract may be 1172E10.

The pathogenic fungi of the genus Mantus may be Xanthomonas oryzae pathovar oryzae or Xanthomonas campestris pathovar vesicatoria.

In one embodiment of the present invention, the composition of the present invention is not limited thereto, but may contain 0.001 to 90 parts by weight, or 0.001 to 70 parts by weight, of the actinomycetes extract according to 100 parts by weight of the total composition.

In one embodiment of the present invention, the composition of the present invention may contain, in addition to the actinomycetes extract 1172E10, other components capable of synergizing the main effect, etc., within the range not impairing the main effect of the present invention have.

Actinomyces is a microorganism that exists in the form of mycelium and sporophyll in soil, plant, animal, river, seawater, and is classified into prokaryote which is close to bacteria, that is, actinomycetes of bacteria. The cell size is similar to that of bacteria, and the cells grow like a mycelium of fungus in thread-like form and form spores at the end. Actinic actinomycetes play an important role in decomposition of various organic matter, especially degradation of organic matter, and also produce antibiotics.

The present invention also relates to a method for controlling Xanthomonas oryzae pathovar oryzae or Xanthomonas campestris pathovar vesicatoria using the above composition. The above-mentioned control method may be a method for controlling the plant pathogens by spraying or applying the composition to a lesion where a blight of rice blast or blight of pepper leaf blight occurs, or a place where it is expected to occur, although the method is not limited thereto.

In one embodiment of the present invention, it was found that the actinomycetes extract inhibited the IC50 values of 1172E10 and 0.320 mg / l and 0.405 mg / l, respectively, for the rice blast fungus and the red pepper fungus.

The present invention also relates to the use of Streptomyces sp .) is extracted with an organic solvent; And extracting the extract from the actinomycetes of SEQ ID NO: 1, which comprises the step of dissolving the organic solvent extract in an alcohol and then carrying out chromatography thereon.

The organic solvent used in the step of extracting with the organic solvent includes but is not limited to acetone, ethyl acetate, ether, chloroform, pentane, hexane, heptane, nonane, decane, benzene, toluene, xylene, methanol, ethanol, And alcoholic beverages. Herein, the term " alcohol " means ethanol produced by fermenting a starch raw material or a saccharide raw material and then distilling it.

The alcohol in which the organic solvent extract is dissolved is not limited thereto, but any one or more selected from the group consisting of methanol, ethanol, butanol, and alcohol may be used, and methanol may be used.

Although the chromatography is not limited thereto, silica gel, reversed phase silica gel, Sephadex, Diaion resins, alumina, activated carbon, magnesium silicate, calcium carbonate, calcium phosphate, magnesium phosphate, magnesia or potassium hydroxide can be used as the adsorbent And Sephadexes can be used. More specifically, high performance liquid chromatography can be performed once more after performing Sephadex gel column chromatography.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1>

1. Strain and culture conditions

Xanthomonas oryzae pv. oryzae KACC10331 (Xoo) and Xanthomonas campestris pv. vesicatoria KACC11127 (Xcv) was obtained from the Korea Agricultural Culture Collection (KACC). Xoo and Xcv were cultured at 28 占 폚 in a medium rich in YPD (1% yeast extract, 2% peptone and 2% dextrose). The strains of the actinomycetes collected from various parts of Korea including Jeju Island, Baekam, Mangyeong River, and Jirisan were preserved in ECUM (Extract Collection of Useful Microorganisms) library of Myongji University and GSS medium (1% soluble starch at pH 7.2) at 28 ℃. (2% glucose, 0.1% beef extract, 0.4% yeast extract, 0.2% NaCl, 0.025% K ₂ HPO ₄ and 0.2% CaCO ₃ ), Bennett's (BN) medium (glucose 1%, yeast extract 0.1% Bacto-peptone 0.2%, beef extract 0.1%) or DYC medium (dextrin 2.5%, dry yeast 1.2%, corn starch liqueur 2%, NaBr 0.1%, CoCl ₂ 0.1% at pH 7.0). Table 1 below shows the information on the antibacterial actinomycetes strain 10146GE and extracts.

2. Preparation of actinomycetes

The actinomycetes strains collected from various regions were cultured in three different culture media (GSS medium, Bennett's medium (BN) medium and DYC medium with poor nutritional content). Three extract fractions were prepared from each actinomycetes culture broth. 30 ml of culture broth was centrifuged at 5000 xg for 10 min. The mycelium extracted with 3 ml of methanol was collected as pellet. The fraction (Mycelia Isolated from Supernatant, MIS) is a mycelium isolated from the supernatant. After centrifugation of the mycelium, the supernatant was further extracted with 30 ml of ethyl acetate and dried. result The dried extract was dissolved in 3 ml of methanol. This is the Secreted Hydrophobic (SHB) fraction. The remaining ethyl acetate-insoluble fraction is the Secreted Hydrophilic (SHL) fraction. All fractions were completely dried until use. The dry weight was measured for the calculation of GI50 or for the dose required to cause 50% growth inhibition.

3. Antibacterial effect test

The antimicrobial effect of the extract was measured by a two - step single - dose growth inhibition test. The first screen preserved all of the extracts in columns 2 to 10 of a 96-well plate, and the columns 1, 11, and 12 were empty (but not infected), bacterial culture and 6% dimethylsulfoxide (DMSO) as controls for bacterial cultures Respectively. Actinomycetes extracts stored in 96-well plates were dissolved in 40 μl of 50% DMSO and diluted further with 100 μl of YPD medium. Xoo and Xcv are the initial exponential growth steps (OD ₆₀₀ = 0.2), and 160 [mu] l of bacterial culture was distributed to each well of a 96-well microplate (Nunc ^TM ). Next, 40 [mu] l of pre-diluted actinomycetes extract was added to each well. Bacterial cells in 96-well microplates were incubated at 28 [deg.] C with stirring at 700 rpm. The OD ₆₀₀ values of each well in the culture dish were measured every 4 hours for 20 hours using a multi-microplate reader (infinite M200 PRO, TECAN). In order to measure the antimicrobial activity of each extract, GIR = (1- ( C - B ) / ( A - B )) x100 at 20 hours in the case of Xoo and Xcv where A is OD ₆₀₀ of bacterial culture with 6% DMSO value, B 0.2, which in cell culture the addition of extract of OD ₆₀₀ value, C, using the OD ₆₀₀ value) of the bacterial culture to the extract, was calculated and the growth inhibition.

For the second growth inhibition screen, the extracts selected for the first analysis were identified three times in the same procedure as for the first analysis.

In contrast to the control, the amount of growth inhibition was measured in five different amounts (1, 10 ^-1 , 10 ^-2 , 10 ^-3 , and 10 ^-4 dilutions, respectively) of the extract. From this, the required amount that resulted in GI50 or 50% growth inhibition was calculated. All experiments were performed three times.

4. Actinomycetes Extract

Actinomycetes strains according to various culturing conditions produce various physiological supplementary metabolites. To produce various supplemental metabolites from actinomycetes strains, media with three different nutrients were used to grow actinomycetes cells. In addition, three extract fractions were prepared for each actinomycetes cultivation. The mycelial fraction was separated from the culture supernatant. The hydrophobic compound (SHB) was extracted from the supernatant fraction by ethyl acetate. The hydrophobic compound was obtained from the residual water layer (SHL). A total of 5,328 extract fractions were generated and investigated for antimicrobial activity against two plant pathogens Xoo and Xcv. Table 2 below is information on 1172E10.

5. First screening for antimicrobial activity

More than 50% cell growth inhibitory activity was considered as an extract of antimicrobial activity; 34 and 29 extracts were selected against Xoo and Xcv, respectively, which constituted 0.64% and 0.53% of all tested extracts (see Figure 1 and Table 3). Table 3 below lists the first and second investigated antimicrobial extracts against Xoo and Xcv.

1172H9, the most effective antimicrobial extract against Xoo, inhibited Xoo cell growth by 90%. Two of the top 34 extracts against Xoo were in SHL, 16 of which were in MIS and 16 were in SHB (Figure 2A). In screening against Xcv, 1131H3 was the most effective and had about 100% of the growth inhibitory effect. Three of the top 29 extracts with growth inhibition against Xcv were in SHL: 10 of them were in MIS and 15 were in SHB (Fig. 2B).

6. Second screening for antimicrobial activity

In order to confirm the antimicrobial activity, an extract showing high growth-inhibiting activity in the first screening was tested three times. 1172E10 (SHB) had the highest growth-inhibitory activity against both Xoo and Xcv (Figures 3A and 3B). 1172E10 (SHB) was collected from the same actinomycetes strain from Jeju Island (see Table 1). The extract of 1172E10 (SHB) was found as an antimicrobial extract against Xoo and Xcv.

The required dose to induce GI50s or 50% growth inhibition in the tested antimicrobial extracts was determined as a dilution series of extracts (Table 4). In Xoo and Xcv, 1172E10 (SHB) showed the highest antimicrobial activity, especially 1172E10 (SHB) showed a GI50 value of ug / ml or less. Table 4 below shows the GI50s of the irradiated antimicrobial extract against Xoo and Xcv.

<110> Foundation of Agri. Tech. Commercialization & Transfer <120> Composition containing extracts of Streptomyces for preventing          Xanthomonas spp. <130> P120808 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1423 <212> DNA <213> Artificial Sequence <220> <223> MJM10146 <400> 1 ggcgggtgct taccatgcag tcgaacgatg aaccacttcg gtggggatta gtgggagaacg 60 ggtgagtaac acgtgggcaa tctgccctgc actctgggac aagccctgga aacggggtct 120 aataccggat actgaccttc acgggcatct gtgaaggtcg aaagctccgg cggtgcagga 180 tgagcccgcg gcctatcagc ttgttggtga ggtaatggct caccaaggcg acgacgggta 240 gccggcctga gagggcgacc ggccacactg ggactgagac acggcccaga ctcctacggg 300 aggcagcagt ggggaatatt gcacaatggg cgaaagcctg atgcagcgac gccgcgtgag 360 ggatgacggc cttcggggtg taaacctctt tcagcaggga agaagcgaaa gtgacggtac 420 ctgcagaaga agcgccggct aactacgtgc cagcagccgc ggtaatacgt agggcgcaag 480 cgttgtccgg gaattattgg gcgtaaagag ctcgtaggcg gcttgtcacg tcggttgtga 540 aagcccgggg cttaaccccg ggtctgcagt cgatacgggc aggctagagt tcggtagggg 600 agatcggaat tcctggtgta gcggtgaaat gcgcagatat caggaggaac accggtggcg 660 aaggcggatc tctgggccga tactgacgct gaggagcgaa agcgtgggga gcgaacagga 720 ttagataccc tggtagtcca cgccgtaaac ggtgggcact aggtgtgggc aacattccac 780 gttgtccgtg ccgcagctaa cgcattaagt gccccgcctg gggagtacgg ccgcaaggct 840 aaaactcaaa ggaattgacg ggggcccgca caagcggcgg agcatgtggc ttaattcgac 900 gcaacgcgaa gaaccttacc aaggcttgac atacaccgga aacgtccaga gatgggcgcc 960 cccttgtggt cggtgtacag gtggtgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1020 gggttaagtc ccgcaacgag cgcaaccctt gtcccgtgtt gccagcaggc ccttgtggtg 1080 ctggggactc acgggagacc gccggggtca actcggagga aggtggggac gacgtcaagt 1140 catcatgccc cttatgtctt gggctgcaca cgtgcctaca atggccggta caatgagctg 1200 cgataccgca aggtggagcg aatctcaaaa agccggtctc agttcggatt ggggtctgca 1260 actcgacccc atgaagtcgg agtcgctagt aatcgcagat cagcattgct gcggtgaata 1320 cgttcccggg ccttgtacac accgcccgtc acgtcacgaa agtcggtaac acccgaagcc 1380 ggtggcccaa ccccttgtgg gagggagctt cgaagtgact gcc 1423

Claims (5)

(Xanthomonas campestris pathovar vesicatoria) containing an extract of Streptomyces sp. MJM10146GE extracted with ethyl acetate as an active ingredient, characterized in that the 16S rRNA gene comprises the nucleotide sequence of SEQ ID NO: / RTI &gt; delete The composition according to claim 1, wherein the actinomycetes extract is contained in an amount of 0.001 to 90 parts by weight based on 100 parts by weight of the total composition. delete A method for controlling Xanthomonas campestris pathovar vesicatoria using the composition according to claim 1 or 3.

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