KR20180105460A - Burkholderia stabilis PG159 strain isolated from mountain-cultivated ginseng roots having antimicrobial activity against ginseng plant pathogen and uses thereof - Google Patents

Abstract

The present invention relates to a Burkholderia stabilis PG159 strain isolated from wood-cultivated ginseng roots and having an antibacterial activity against a ginseng plant pathogen, and a use thereof. According to the present invention, it has been confirmed that a Burkholderia stabilis PG159 strain, which is endogenous bacteria isolated from wild wood-cultivated ginseng roots, inhibits growth of a main ginseng pathogen. The Burkholderia stabilis PG159 strain of the present invention is safe for environmental ecosystems, has no drug resistance appearance, can be used as a bio-agricultural chemical for securing safety of agricultural products, and can be utilized in a wood-cultivated ginseng cultivation site through an environmentally-friendly control of major pathogens which are most problematic when cultivating a wood-cultivated ginseng. Thus, the Burkholderia stabilis PG159 strain is very useful in related industries.

Description

BURCHHOLDERIA STABILIS PG159 strain having antimicrobial activity against ginseng plant pathogenic bacteria isolated from the root of ginseng and its use {Burkholderia stabilis PG159 strain isolated from a mountain ginseng root having antimicrobial activity against ginseng plant pathogen and uses thereof}

The present invention relates to a strain of Burkholderia stablis PG159 having antimicrobial activity against ginseng plant pathogenic bacteria isolated from goat's roots and its use.

The major diseases that occur during the cultivation of goat ginseng are the soil infectious pathogens Cylindrocarpon ( Cylindrocarpon) destructans ), Rhizoctonia solani , Botrytis cinerea ), Phytophthora ( Phytophthora) cactorum ) and Alternaria panax ), and is causing damage to most of the goat farms nationwide. Especially, 4 ~ 7% occurred in 4 ~ 6 year old, and 12% in 4 year old spore. In other words, the cause of the high birth rate during the cultivation of the goat ginseng is caused by the melancholy. However, it is very urgent to develop eco-friendly pollution control technology because it is difficult to solve the problem of high birth rate due to failure of selection of pollution control agent,

One of the alternative methods of safer and less harmful pesticides is biopesticides. The subject of biological pesticides can be classified as using microorganisms (fungi, bacteria, viruses) and active substances produced by microorganisms, which are the resources themselves, which are the resources that exist in nature, and their application fields are sterilization, insecticide, And an anti-injury agent have been put to practical use.

The importance of environmentally friendly bio-pesticides has been highlighted and the usage of bio-pesticides has been rising. The global bio-pesticide market is expected to increase rapidly in the future, and the domestic market is expected to grow to more than 180 billion won And the global market size is estimated to reach US $ 5 billion by 2015, about 15% of the pesticide market.

Microorganisms have been found to have a wide variety of morphological and habitat environments, and the chemical structures of the metabolites they produce are also very diverse. Suggesting that various chemical structures have various physiological activities. Regarding the metabolites produced by the fungus, in 1929 Alexander Clamming discovered that Penicillium Many antibiotics have been found in microorganisms such as bacteria, actinomycetes, and fungi since the discovery of penicillin in notatum cultures. In the case of metabolites derived from plants, there are several times when the sample is secured. However, since microorganisms can be cultured, once the culture method is established, mass production can be advantageous.

Endophyte, a plant symbiotic, is known to protect plants against various stresses. However, there is almost no research on endogenous microorganisms in ginseng, an important medicinal plant. It is necessary to develop eco - friendly control technology to isolate various endogenous microorganisms from the non - pesticide - free goat ginseng and inhibit the major pathogenic microorganisms that cause large - scale damage during the cultivation of the goat ginseng.

 Korean Patent No. 0312731 discloses a use of a novel strain of Bacillus subtilis for the prevention of ginseng side-effects, and in Korean Patent No. 1531322, there is disclosed an anti-fungal activity against ginseng root rot bacteria A novel strain of Bacillus subtilis AM-1058 and its culture medium composition is disclosed. However, as in the present invention, a strain of Burkholderia strains PG159 having antimicrobial activity against ginseng plant pathogenic bacteria isolated from the root of goatsam Application "has not been disclosed at all.

The present invention has been made in view of the above-mentioned needs. In the present invention, to find endogenous bacterium having antimicrobial activity against major pathogens occurring in the cultivation of goat ginseng, 240 goat gins were collected from 9 sites and 24 sites nationwide, As a result of isolating and identifying the endogenous bacteria from the root, stem and leaf tissues of the goat ginseng, it was confirmed that the Bactholderia stavilis PG159 strain has a very excellent antimicrobial effect against the six causative pathogens showing serious virulence in the ginseng plant, .

In order to accomplish the object of the present invention, the present invention provides a method for producing a ginseng plant pathogenic bacterium, which comprises the step of contacting Burkholderia stabilis ) strain.

The present invention also provides a microorganism preparation for antimicrobial activity against a causative agent of ginseng plant disease comprising the strain or a culture solution thereof as an active ingredient.

The present invention also provides a method for controlling a ginseng plant disease comprising treating the effective amount of the strain or a culture thereof with a ginseng plant, a seed of a ginseng plant, or a ginseng cultivation plant.

In the present invention, endogenous bacteria isolated from goat's roots, Burkholderia stabilis PG159 inhibited the growth of major ginseng pathogens. Burke holde Ria star of the present invention's borrowing (Burkholderia stabilis ) PG159 strain is safe for environmental ecosystem, it can be used as a biochemical pesticide which does not show resistance to chemicals and secures the safety of agricultural products. It is applied to the goat ginseng cultivation area through environmentally friendly control of major pathogens Possibly, it is very useful for related industries.

Brief Description of the Drawings Fig. 1 is a graph showing the results of the endogenous bacterium Burkholderia stabilis PG159 was subcultured on TSA plate medium to confirm that it was a pure culture.
FIG. 2 is a graph showing the effect of the endogenous bacterium Burkholderia < RTI ID = 0.0 > stabilis) the result shows the 16S rRNA base sequence of PG159 strain.
FIG. 3 is a graph showing the effect of the endogenous bacterium Burkholderia < RTI ID = 0.0 > ( Burkholderia < / RTI > stabilis ) PG159 strain of the present invention.
FIG. 4 is a graph showing the effect of the endogenous bacterium Burkholderia stabilis ) PG159 strain in the growth inhibitory activity against ginseng pathogens. The ginseng pathogens are, in order from the left, Alternaria panax , A), Cylindrocarpon destructans ( B), Rhizoctonia solani , C), Phytophthora ( Phytophthora) cactorum , D) and Pythium sp., E).
Figure 5 holde Leah Burke stars in Billy's Ginseng (Burkholderia stabilis ) PG159 to ginseng toxicity and viability.

In order to accomplish the object of the present invention, the present invention provides a method for producing a ginseng plant pathogenic bacterium, which comprises the step of contacting Burkholderia stabilis ) strain.

In the strain in accordance with an embodiment of the invention, said strain is the buck holde Ria star accession number KCTC13218BP Billy's (Burkholderia stabilis PG159 strain, but is not limited thereto.

In order to find endogenous bacteria having antimicrobial activity against major pathogens occurring during the cultivation of goat ginseng in the present invention, 240 goat gins were collected from 9 sites and 24 sites in the whole country, and all the endogenous germs were collected from root, stem, The genomic DNA was isolated and identified as Burkholderia stabilis as a result of 16S rDNA sequence analysis. The above-mentioned strains were found to have excellent antimicrobial effect against six causative bacteria showing serious pathogenicity in ginseng plants. Thus, the strain Burke holde Ria star Billy's (Burkholderia stabilis PG159, deposited at the Korea Biotechnology Research Institute on Feb. 17, 2017 (Deposit No. KCTC13218BP).

In a strain according to an embodiment of the present invention, the causative agent of ginseng plant disease is selected from the group consisting of Alternaria panax , Botrytis < RTI ID = 0.0 > cinerea , Cylindrocarpon destructans , Rhizoctonia solani ), Phytophthora ( Phytophthora) cactorum ) and Pythium sp.), but the present invention is not limited thereto.

The present invention also provides a microorganism preparation for antimicrobial activity against a causative agent of ginseng plant disease comprising the strain or a culture solution thereof as an active ingredient.

The antimicrobial microbial preparation for ginseng plant pathogenic bacteria of the present invention may be in the form of a wettable powder or a suspension concentrate. The antimicrobial microbial preparation according to the present invention may be prepared in the form of a liquid sterilizing agent, and may be added in the form of a powdered powder, or may be granulated by formulating it into a powder. However, the formulation is not particularly limited. In other words, it can be formulated as a bio-disinfectant to overcome this in environment-friendly organic farming with limited supply of chemical disinfectants.

Wettable powder of the present invention as an active ingredient (active ingredient) Burke holde Ria star Billy's (Burkholderia stabilis PG159 strain or culture thereof, white carbon as a moisture absorbent, sodium bis [2-ethylhexyl] sulfosuccinate as a wetting agent, sodium lignosulfonate as a dispersing agent, and kaolin as an extender, 10% by weight of star Billy Burke holde Leah's (Burkholderia stabilis PG159 strain or culture thereof, 1 wt% white carbon, 1 wt% sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium lignosulfonate and 87 wt% kaolin But is not limited thereto.

Liquid wettable powder of the present invention, the active ingredient (active ingredient) as a buck holde Ria star Billy's (Burkholderia stabilis PG159 or its culture, wetting agent and MBSC (nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, sodium bis [20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol) as a wetting agent and dispersant, isopropanol as an excipient and water as an extender. The liquid wettable agent is preferably present in an amount of from 50% by weight to < RTI ID = 0.0 > Burkholderia & stabilis PG159 strain or culture thereof, 4 wt% MBSC, 30 wt% isopropanol and 16 wt% water, but is not limited thereto.

The present invention also provides a method for controlling a ginseng plant disease comprising treating the effective amount of the strain or a culture thereof with a ginseng plant, a seed of a ginseng plant, or a ginseng cultivation plant.

The ginseng botanical bottle may be selected from the group consisting of Alternaria panax , Botrytis < RTI ID = 0.0 > cinerea , Cylindrocarpon destructans , Rhizoctonia < RTI ID = 0.0 > solani ), Phytophthora ( Phytophthora) cactorum ) and Pythium sp., But the present invention is not limited thereto.

As a method for controlling the ginseng plant disease, the Burkholderia stabilis ) The effective amount of the culture medium in which the PG159 strain has been cultured can be treated by treating the ginseng plant, the seed of the ginseng plant, or the ginseng plantation.

The 'effective amount' of the present invention is an amount sufficient to cause beneficial or desired results. The microbial agent having antimicrobial activity against ginseng plant pathogens is uniformly diluted with water to control the ginseng plant disease, It can be applied to the ginseng plant and ginseng cultivated land using the apparatus. When the wettable powder or the liquid wettable powder of the present invention is diluted in water, the concentration of the wettable powder or the liquid wettable powder is adjusted to about 10 ⁵ to 10 ¹⁰ cfu / ml, preferably about 10 ⁸ cfu / ml so that the effective ingredient can be in a biologically effective range But is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  1. Burke Hodderia Stabelis ( Burkholderia stabilization ) PG159  Isolation of strain

In order to find endogenous bacterium having antimicrobial activity against major pathogens occurring during the cultivation of goat ginseng, 240 goat gins were obtained from 9 regions and 24 regions in Korea. After cutting the roots, stems and leaves of the obtained goat ginseng, the following disinfection process (immersion in 75% ethanol for 1 minute, immersion in 2% sodium hypochlorite for 10 minutes, immersion in 75% ethanol for 30 seconds, 3 times for 1 minute, dried on sterilized tissue paper), and then plated on TSA (tryptic soy agar) plate medium and cultured at 28 ° C. All endogenous bacteria that grew up to 1 ~ 3 days after dentin were isolated.

Example  2. Burke Hodderia Stabelis ( Burkholderia stabilization ) PG159  Identification of the strain

As shown in FIG. 1, the genomic DNA was isolated to analyze the 16S rDNA nucleotide sequence in order to carry out the molecular biology identification of the endogenous bacteria isolated pure. Burkholderia stabilis) the genomic DNA of the strain PG159 was extracted using the DNeasy Plant Mini Kit (Qiagen, Valencia , CA, USA). For the sequencing analysis, the primers shown in Table 1 below, which target the regions V1 to V3 of 16S rDNA, were used. As a result, as shown in Fig. 2, the 16S rDNA sequence (SEQ ID NO: 1) was identified as Burkholderia stabilis through BLAST search of NCBI. Thus, the strain Burke holde Ria star Billy's PG159 (Burkholderia stabilis PG159) was named strain (Fig. 3).

The primers used in the present invention Name of the primer The forward direction (5'-3 ')
Primer base sequence Name of the primer The reverse direction (5'-3 ')
Primer base sequence 27F AGAGTTTGATCMTGGCTCAG (SEQ ID NO: 2) 1492R GGYTACCTTGTTACGACTT (SEQ ID NO: 3)

Example  3. Burke Hodderia Stabelis ( Burkholderia stabilization ) PG159  Of ginseng pathogen Road drag  Confirm

In order to confirm the antibacterial activity of the strain Burkholderia stabilis PG159 isolated and identified in Examples 1 and 2 against ginseng pathogens, the ginseng pathogen Alternaria panax ) KACC 44351, Botrytis cinerea ) KACC 43521, Cylindrocarpon destructans KACC 44656, Rhizoctonia solani ) KACC 40123, Phytophthora ( Phytophthora) cactorum ) KACC 40166 and Pythium sp.) KACC 40581 were subjected to the following experiment.

In order to verify the antimicrobial activity, Burkholderia stablis strain PG159 (OD ₆₀₀ = 0.5) and six ginseng pathogens were placed at 40 mm intervals on both sides of a tryptic soy agar (TSA) plate medium. At this time, after culturing for 3 to 7 days at 25 ° C, which is the growth temperature of various pathogens, the diameters of pathogenic bacteria were measured and the antibacterial activity was investigated using the following equation.

Inhibition rate of pathogenic growth (%) = [(Growth length of untreated soil - Growth length of treated soil) / Growth length of untreated soil] x 100.

As a result, as shown in Fig. 4, it was confirmed that the Burkholderia stablis PG159 strain of the present invention exhibited a strong antibacterial activity against ginseng pathogens.

Example  4. Burke Hodderia Stabelis ( Burkholderia stabilization ) PG159  Identification of toxicity and viability of ginseng to the strain

To confirm the toxicity test and survival ability of Burkholderia stabilis strain PG159 isolated and identified in Examples 1 and 2 to ginseng, the 2-year-old seedling was purchased from Jinshan, The surface was sterilized in the same manner. The sterilized seeds were transferred to Burkholderia stabilis ) After immersing in a suspension of strain PG159 (0.5 OD ₆₀₀ ) for 40 minutes, the surface was dried and cultivated in sterilized ginseng soil (sacred minerals) in a plant culture incubator (50 ± 10 μmol m ^-2 , 16 h photoperiod) (Fig. 5A). After 2 weeks, the ginseng roots were separated to confirm the presence or absence of pathology (Fig. 5B). The surface of the bacterium was resterilized after surface sterilization and the degree of clustering was calculated using the following equation.

Colonization frequency = (total segment formed by colony) / (total segment) * 100.

As a result, Burkholderia stablis PG159 strain was found to be not harmful to ginseng and a very high clustering degree of 60% in the roots (Fig. 5C).

Korea Biotechnology Research Institute KCTC13218BP 20170217

<110> Research Cooperation Foundation of Yeungnam University <120> Burkholderia stabilis PG159 strain isolated from          mountain-cultivated ginseng roots having antimicrobial activity          against ginseng plant pathogen and uses thereof <130> PN17073 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 1391 <212> DNA <213> Burkholderia stabilis <400> 1 agtcgaacgg cagcacgggt gcttgcacct ggtggcgagt ggcgaacggg tgagtaatac 60 atcggaacat gtcctgtagt gggggatagc ccggcgaaag ccggattaat accgcatacg 120 atctacggat gaaagcgggg gaccttcggg cctcgcgcta tagggttggc cgatggctga 180 ttagctagtt ggtggggtaa aggcctacca aggcgacgat cagtagctgg tctgagagga 240 cgaccagcca cactgggact gagacacggc ccagactcct acgggaggca gcagtgggga 300 attttggaca atgggcgaaa gcctgatcca gcaatgccgc gtgtgtgaag aaggccttcg 360 ggttgtaaag cacttttgtc cggaaagaaa tccttggctc taatacagtc gggggatgac 420 ggtaccggaa gaataagcac cggctaacta cgtgccagca gccgcggtaa tacgtagggt 480 gcgagcgtta atcggaatta ctgggcgtaa agcgtgcgca ggcggtttgc taagaccgat 540 gtgaaatccc cgggctcaac ctgggaactg cattggtgac tggcaggcta gagtatggca 600 gaggggggta gaattccacg tgtagcagtg aaatgcgtag agatgtggag gaataccgat 660 ggcgaaggca gccccctggg ccaatactga cgctcatgca cgaaagcgtg gggagcaaac 720 aggattagat accctggtag tccacgccct aaacgatgtc aactagttgt tggggattca 780 tttccttagt aacgtagcta acgcgtgaag ttgaccgcct ggggagtacg gtcgcaagat 840 taaaactcaa aggaattgac ggggacccgc acaagcggtg gatgatgtgg attaattcga 900 tgcaacgcga aaaaccttac ctacccttga catggtcgga atcccgctga gaggtgggag 960 tgctcgaaag agaaccgata cacaggtgct gcatggctgt cgtcagctcg tgtcgtgaga 1020 tgttgggtta agtcccgcaa cgagcgcaac ccttgtcctt agttgctacg caagagcact 1080 ctaaggagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcctcatggc 1140 ccttatgggt agggcttcac acgtcataca atggtcggaa cagagggttg ccaacccgcg 1200 agggggagct aatcccagaa aaccgatcgt agtccggatt gcactctgca actcgagtgc 1260 atgaagctgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggt 1320 cttgtacaca ccgcccgtca caccatggga gtgggtttta ccagaagtgg ctagtctaac 1380 cgcaaggagg a 1391 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tccgtaggtg aacctgcgg 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tcctccgctt attgatatgc 20

Claims (6)

Burke holde with antibacterial activity against ginseng plant disease organisms Leah star Billy's (Burkholderia stabilis ) strain. The method of claim 1 wherein the strain is the buck holde Ria star accession number KCTC13218BP Billy's (Burkholderia stabilis PG159 strain. The method according to claim 1, wherein the ginseng plant pathogenic bacteria is selected from the group consisting of Alternaria panax , Botrytis &lt; RTI ID = 0.0 &gt; cinerea , Cylindrocarpon destructans , Rhizoctonia solani ), Phytophthora ( Phytophthora) cactorum ) and Pythium wherein the strain is at least one strain selected from the group consisting of sp. A microorganism preparation for antimicrobial use for a causative agent of ginseng plant disease comprising the strain of any one of claims 1 to 3 or a culture thereof as an active ingredient. A method for controlling a ginseng plant disease comprising the step of treating an effective amount of the strain of any one of claims 1 to 3 or a culture thereof with a ginseng plant, a seed of ginseng plant or a ginseng plantation. 6. The plant of claim 5, wherein the plant disease is selected from the group consisting of Alternaria panax , Botrytis &lt; RTI ID = 0.0 &gt; cinerea , Cylindrocarpon destructans , Rhizoctonia &lt; RTI ID = 0.0 &gt; solani ), Phytophthora ( Phytophthora) cactorum ) and Wherein the disease is caused by at least one ginseng causative agent selected from the group consisting of Pythium sp.

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