KR20200078168A - Pseudomonas frederiksbergensis strain possessing antifungal activity against major pathogenic bacteria of plant and use thereof - Google Patents

Abstract

The present invention relates to a Pseudomonas frederiksbergensis strain having antibacterial activity against major plant pathogens. In addition, the present invention relates to a composition for controlling plant pathogens comprising anyone or more selected from the group consisting of the strain, a culture solution thereof, and a mixture thereof, to a method for controlling plant pathogens comprising a step of treating the composition.

Description

Pseudomonas frederiksbergensis strain possessing antifungal activity against major pathogenic bacteria of plant and use thereof}

The present invention relates to a Pseudomonas frederiksbergensis strain having antimicrobial activity against major plant pathogens.

In addition, the present invention relates to a plant pathogen control method comprising the step of treating a composition for controlling a plant pathogen comprising at least one selected from the group consisting of the strain, a culture medium thereof, and a mixture thereof.

Root rot on ginseng causes root rot in low temperature at 18-21℃ and neutral soil at pH 7.0, which is accompanied by softness when soil moisture is high. In the process of cultivating ginseng, it exhibits redness symptoms on the surface of the roots and deteriorates the marketability. In fact, it is common for seedlings planted at farms to show a residual rate of about 50% or less at harvest after 5 years. The reason is that root pathogens inhabit the roots of ginseng during the initial cultivation. As a method for controlling ginseng root rot, currently, biological control methods using antagonistic microorganisms, and chemical control methods using soil fumigants such as cylon and bassamide in the case of arable land, and rice field cultivation methods in field reptiles are used.

The gray mold rot that causes root rot of ginseng is a pathogen by Botrytis cinerea , a type of incomplete fungus, which is a very versatile strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin Causes rot on almost all vegetables and fruits, such as cabbage, lettuce, asparagus, potatoes, kidney beans, and peas. The gray mold disease can be controlled by controlling using resistant varieties, controlling the cultivation environment and seedling control by removing diseased plants, chemical control using fungicides, or biological control using microorganisms, but mainly depends on chemical control.

However, the above existing control methods for soil infectious pathogens do not have a distinct effect, and in particular, the chemical control method does not have a long-lasting effect and destroys soil ecosystem due to resistance bacteria, toxicity, or chemicals remain in the plant and ingest it as a result. It may be toxic to mammals. Therefore, for effective soil disease control, biological control is most suitable as a control measure for ginseng disease, which is effective and stable in the environment due to the nature of ginseng cultivation, but lacks understanding of various root-soil soil environments and does not have high antibacterial or antibacterial activity against selected pathogens. No antagonistic microorganisms with excellent control effect against ginseng root rot disease such as poor ability to settle bacteria in soil or seedling due to lack of preparation method or treatment method have not been found.

Endophytic bacteria inhabit and interact with plants without causing disease symptoms. They are ubiquitous in plant species and inhabit a variety of plant tissues and can have beneficial effects such as plant growth, suppressing plant pathogens, and improving plant nutrition and antibiotic production. Because of the stable habitat provided by the host plant, endogenous bacteria are less competitive with nutrients and are protected from various external stresses. Therefore, the bacteria can exert more consistent effects than the phyllosphere or rhizosphere bacteria that exist outside the plant.

Republic of Korea Patent Publication No. 10-2018-0116172 is for buckholeteria ambiparia CJ4 and its use having antibacterial activity against ginseng root rot pathogens, and buckholeteria ambipariah CJ4 is for various ginseng root rot pathogens It has been disclosed to have a high antibacterial effect, such as suppressing live bacteria. However, no research or description has been disclosed on whether Pseudomonas fredericksbergensis of the present invention among ginseng endogenous bacteria has an antibacterial effect against a major ginseng pathogen.

Accordingly, the present inventors have tried diligently to provide a biological control agent that can be used as a control agent for root rot or gray mold disease causative agents of plants, particularly ginseng, a new Pseudomonas frederiksbergensis among ginseng endogenous strains. ) As a strain, it was confirmed that the Pseudomonas fredericksbergensis PgBE45 strain exhibited antibacterial activity against major pathogens of ginseng and completed the present invention.

Accordingly, it is an object of the present invention to provide a Pseudomonas frederiksbergensis strain having antibacterial activity against major pathogens of plants, particularly ginseng.

Another object of the present invention is to provide a plant pathogen control method comprising the step of treating a composition for controlling plant pathogens comprising at least one selected from the group consisting of the strain, a culture medium thereof, and mixtures thereof.

In order to achieve the above object, the present invention is Cylindrocarpon destructans ( Cylindrocarpon destructans ), Botrytis Cinerea , Phytophthora cactorum , Pythium ultimum , Alta or Ria Panax (Alternaria panax), Fusarium solani (Fusarium solani) and Rhizoctonia small Raney Pseudomonas Fredericks Bergen with antibacterial activity against one or more pathogens selected from the group consisting of (Rhizoctonia solani). system (Pseudomonas frederiksbergensis ) strain.

According to a preferred embodiment of the present invention, the pathogen may cause root rot or gray mold on the plant.

According to a preferred embodiment of the present invention, the root rot disease may be from any one or more plants selected from the group consisting of ginseng, strawberry, potato, lily, walnut, orange and olive.

According to a preferred embodiment of the present invention, the gray mold disease is any one or more plants selected from the group consisting of ginseng, strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin, cabbage, lettuce, asparagus, potato, kidney beans and peas It may be onset.

According to a preferred embodiment of the present invention, the strain may include the 16S rRNA nucleotide sequence of SEQ ID NO: 3.

According to a preferred embodiment of the present invention, the strain may be a strain endogenous at any one or more sites selected from the group consisting of ginseng root, stem, leaf, flower, flower stem and outpost.

According to a preferred embodiment of the present invention, the strain may be Pseudomonas Fredericks Bergensis PgBE45 strain of accession number KACC 92246P.

The present invention also provides a composition for controlling plant pathogens comprising any one or more selected from the group consisting of strains, cultures thereof, and mixtures thereof.

According to a preferred embodiment of the present invention, the plant may be ginseng (ginseng).

The present invention also provides a method for controlling plant pathogens comprising the step of treating the composition for control.

According to a preferred embodiment of the present invention, the plant may be ginseng (ginseng).

Pseudomonas fredericksbergensis strain has excellent antimicrobial activity against pathogens of plants, especially pathogens causing root rot or gray mold on ginseng, and thus can be effectively used in a composition or method for controlling plant pathogens. In particular, the above effect can be obtained most significantly by Pseudomonas Fredericks Bergensis PgBE45 (Accession Number: KACC 92246P) of the present invention.

Figure 1 shows the results of the classification according to the age of the ginseng collected from the distribution or diversity of the obtained ginseng endogenous strains. a) is the neck, b) is the phylum, c) is the result of classifying endogenous strains according to the Shannon diversity index, d) is the endogenous commonly found in ginseng by age group Shows the number of strains.
Figure 2 shows the results classified according to the portion of ginseng collected from the distribution or diversity of endogenous strains of ginseng. a) is the neck, b) is the phylum, c) is the result of classifying endogenous strains according to the Shannon diversity index, d) is the endogenous commonly found in each part of ginseng. Shows the number of strains.
Figure 3 shows the results of in vitro antagonism analysis. DW, OD ₆₀₀ of 0.1, PgBE45 of 0.1 and PgBE45 of OD ₆₀₀ of 0.01 and ginseng major pathogens (C. destructans or B.cinerea) were treated at positions 1 to 4 of the plate, and the antibacterial effect was confirmed. PgBE45 of the present invention showed a significant antibacterial effect against ginseng major pathogens.

Hereinafter, the present invention will be described in more detail.

As described above, in the prior art, a chemical control method has been mainly used to control root rot or gray mold disease of plants, especially ginseng, but the effect is not persistent, and resistant bacteria are generated, toxic to soil ecosystem or to plants due to toxicity As a result, it has a problem of remaining toxic to the mammal that ingests it. Biological control methods have also been studied, but their effects have been negligible.

Pseudomonas frederiksbergensis strain according to the present invention, in particular Pseudomonas frederiksbergensis strain PgBE45 is environmentally friendly as a ginseng endogenous strain and has an antimicrobial effect on ginseng main pathogens, so it is effective in the composition or control method for controlling plant pathogens Can be used. At this time, the "plant pathogen" of the present invention means a causative agent causing disease in plants.

Accordingly, the present invention is Cylindrocarpon destructans ), Botrytis Cinerea , Phytophthora cactorum ), Pythium ultimum ), Alternaria panax , Fusarium solani) and Rhizoctonia small Raney (Rhizoctonia solani) group which Pseudomonas Fredericks Bergen sheath (Pseudomonas frederiksbergensis with antibacterial activity against one or more pathogens selected from the consisting of a) it is possible to provide a strain.

The pathogen of the present invention is preferred to cause root rot or gray mold disease in plants, but is not limited thereto.

The root rot of the present invention is preferably from any one or more plants selected from the group consisting of ginseng, strawberry, potato, lily, walnut, orange and olive.

The gray mold disease of the present invention is preferably from any one or more plants selected from the group consisting of ginseng, strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin, cabbage, lettuce, asparagus, potato, kidney beans and peas, More preferably, it is preferred to develop ginseng.

The strain of the present invention preferably includes the 16S rRNA nucleotide sequence of SEQ ID NO: 3, more preferably Pseudomonas Fredericks Bergensis PgBE45 strain of accession number KACC 92246P.

The strain of the present invention is preferably a strain endogenous at any one or more sites selected from the group consisting of roots, stems, leaves, flowers, flower stems and outposts of ginseng, more preferably roots, stems and leaves of ginseng It is preferably a strain endogenous at any one or more sites selected from the group consisting of. The ginseng is preferably Panax ginseng , but is not limited thereto.

In addition, the present invention can provide a composition for controlling plant pathogens comprising any one or more selected from the group consisting of the above-described strain, a culture solution thereof, and mixtures thereof.

The culture solution is obtained by inoculating spores of a certain concentration in a medium suitable for microbial growth, and then inducing microorganisms to grow for a certain period in the incubator, a mixture of cells, substances secreted while the cells grow and nutrients necessary for growth. As, it means that it is manufactured through a process of filtering impurities. Specifically, the Pseudomonas fredericksbergensis strain of the present invention is LB (Luria Bertani) medium, nutrient broth (NB) medium, brain heart infusion (BHI) medium, King's medium B (KB) or Tryptic Soy Broth (TSB) medium. It is preferable to mean that it is prepared by culturing in any one or more media selected from the group consisting of, but is not limited thereto.

The plant of the present invention is preferably ginseng (ginseng), but is not limited thereto.

The plant pathogen is Pseudomonas of the present invention ( Pseudomonas frederiksbergensis ) The strain is the same as the pathogen having antimicrobial activity, so the description is substituted.

The control composition is not particularly limited as long as it is a common control agent used for the prevention, reduction or eradication of diseases caused by plant pathogens, and the prevention or reduction is a statistically significant difference with respect to host plants in which the composition is not treated. It means that there is. Preferably, microbial pesticides, seed coatings, microbial pesticides for controlling root rot or gray mold disease of ginseng, additives for the production of non-sterile medium for cultivation of ginseng, bio-soil, microbial nutrients, soil improvers, composting agents, foliar sprayers or irrigation sprays It may be any one or more selected from the group consisting of zero. In particular, Cylindrocarpon , a ginseng root rot pathogen, Cylindrocarpon destructans ) and Botrytis Cinerea have excellent effects on growth inhibition, which can be used for the production of bio-soil, as an additive for the production of non-sterile medium for ginseng cultivation, as a microbial pesticide for controlling ginseng root rot disease. . The microbial pesticide is not particularly limited as long as it is produced by a conventional method, including the Pseudomonas prederix bergensis strain of the present invention. For example, it may include a Pseudomonas fredericksbergensis strain and a bio-matrix that is a microbial carrier, and the bio-matrix may include a plant resource polymer material, a microbial biopolymer material, a nutritional agent, an auxiliary material and an additive. In addition, the plant material polymer material may include potato flour, soybean flour, starch powder, etc., and the microbial origin biopolymer may include mucopolysaccharide, poly-betahydroxy-alkanoate, and flocculant. Further, the nutrient may include glucose, peptone and inorganic salts, the auxiliary material may include vermiculite, rice husk, etc., and the additive may include a bulking agent, a UV blocking agent, and a surfactant. The microbial pesticide may be used as an antifungal microbial pesticide, seed coating agent, microbial nutrition agent, soil conditioner, composting agent, foliar sprayer, or irrigation sprayer.

In addition, the present invention can provide a method for controlling plant pathogens comprising the step of treating the composition for control.

The plant of the present invention is preferably ginseng (ginseng), but is not limited thereto.

The control method of the present invention may be a method for controlling ginseng root rot or gray mold disease by treating the control composition on soil, plants or plant seeds. At this time, the treatment method is a method that is generally performed, that is, spraying (eg spraying, misting, atomizing, powder spraying, granulating spraying, sleep application, regular application, etc.), soil application (eg mixing, irrigation, etc.) , Surface use (eg, coating, smearing, coating, etc.), immersion, poisoning, smoke application. The amount used can be appropriately determined according to the formulation, the damage situation, the application method, and the application location. In the present invention, the effective amount of microorganisms contained in the preparation treated according to the above method may be included in the number of microorganisms of 1 to 1×10 ¹⁰⁰ per arable land area (m 2 ). In addition, among the above methods, the effective amount of microorganisms contained in the formulation treated by spraying may be included at a concentration of 1 to 1×10 ¹⁰⁰ microorganisms per ml, and the effective amount of microorganisms contained in the composition treated by immersion is 1 per ml. To 1×10 ¹⁰⁰ .

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Endogenous strain detach

The endogenous bacteria of the ginseng were isolated, and the distribution diversity of the endogenous strains according to the age of the collected ginseng and the part of the ginseng was examined.

Specifically, 2 to 6 years old native ginseng is collected from the Horticultural and Herbal Science Research Institute (127°45'13.14"E, 36°56'36.63"N) of the Chungbuk Rural Development Administration, and the root, stem, leaf and flower stem parts Separated. The soil on the tissue surface was removed by washing with tap water. The sample was sterilized with 70% ethanol for 1 minute and 12% NaOCl for 3 minutes, and then rinsed 3 times with sterile water. This was transferred to a tube and homogenized by mixing 500 mg of each sample with 1 ml of sterile water using a tissue lyser. 100 μl of each suspension diluted with 10 ⁰ , 10 ^-1 , 10 ^-2 and 10 ^-3 was spread on a Luria-Bertani agar plate and incubated at room temperature for 2 days. Single colonies were isolated and incubated at 28° C. and 200 rpm for 12 hours and then stored at −80° C. until use.

As a result, a total of 42 strains of 116 strains were isolated and classified according to the age and site of the collected ginseng, respectively, and shown in [FIG. 1] and [FIG. 2]. Of the 116 strains 34 strains (30%) of Bacillus (Bacillus) was spp, the highest diversity index Shannon (Shannon diversity index) of 2 years old ginseng and ginseng root. There were two endogenous strains found in ginseng aged 2 to 6 years old, and four endogenous strains found in all parts of ginseng. Among them, PgBE45 of the present invention was commonly isolated from leaves, stems and roots of 5-year-old ginseng.

16S rRNA analysis

Amplify 16S rDNA for bacterial identification and universal bacterial 16S rRNA primer 518F (5'-CCA GCA GCC GCG GTA ATA CG-3', SEQ ID NO: 1) and 800R (5'-TAC CAG GGT ATC TAA TCC -3', Sequences were determined using SEQ ID NO: 2). The 16S rDNA sequence was compared to the corresponding reference sequence retrieved from the GenBank database by BLAST search.

As a result, the 16S rDNA nucleotide sequence of 116 ginseng endogenous strains isolated in [Example 1] was deposited with GenBank under accession numbers MH144224-MH144340. Among them, PgBE45 (MH144268, SEQ ID NO: 3) of the present invention was confirmed to be Pseudomonas frederiksbergensis species.

In vitro antagonistic analysis

The purpose of this study was to determine whether the 116 ginseng endogenous strains isolated from [Example 1] have antifungal activity against major ginseng pathogens.

Specifically, Cylindrocarpon destructans ; C. destructans ) Mycelium was incubated for 1 week at 20℃, and Botrytis cinerea ; B. cinerea ) Mycelium was cultured at 28°C for 3 days. Each of the above pathogens was inoculated in the center of a potato dextrose agar plate. After 2 or 3 days, 10 μl of a bacterial suspension with OD ₆₀₀ = 1.0 or 0.1 on the left and right sides of the plate was treated, respectively. Images were obtained 3 days after co-cultivation of pathogens and endogenous strains.

As a result, a total of 26 strains against C. destructans and a total of 7 strains against B. cinerea showed antifungal activity. Among them, PgBE45 of the present invention shows C. destructans as shown in FIG. 3. And B. cinerea .

[Statistics analysis]

Tukey's honest significant difference test was used to compare the mean Shannon index. All statistics were analyzed using SPSS v.18 software (IBM, Armonk, NY, USA).

[Trust agency]

Depository name: Institute for Agricultural Biotechnology

Accession number: KACC92246

Date of Deposit: 20181023

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Claims (11)

Cylindrocarpon destructans , Botrytis Cinerea , Phytophthora cactorum ), Pythium ultimum , Alternaria Pseudomonas frederiksbergensis strain having antibacterial activity against any one or more pathogens selected from the group consisting of panax ), Fusarium solani and Rhizoctonia solani .
The Pseudomonas frederiksbergensis strain according to claim 1, wherein the pathogen causes root rot or gray mold on plants.
3. The Pseudomonas frederiksbergensis strain according to claim 2, wherein the root rot disease occurs in any one or more plants selected from the group consisting of ginseng, strawberry, potato, lily, walnut, orange and olive. .
The method of claim 2, wherein the gray mold disease occurs in any one or more plants selected from the group consisting of ginseng, strawberry, tomato, eggplant, bell pepper, cucumber, pumpkin, cabbage, lettuce, asparagus, potato, kidney beans, and peas. Characterized by Pseudomonas frederiksbergensis strain.
The Pseudomonas frederiksbergensis strain according to claim 1, wherein the strain comprises the 16S rRNA nucleotide sequence of SEQ ID NO: 3.
The method of claim 1, wherein the strain is Pseudomonas frederiksbergensis characterized in that the strain is endogenous at any one or more sites selected from the group consisting of roots, stems, leaves, flowers, flower stems and outposts of ginseng. ) Strain.
The Pseudomonas frederiksbergensis strain according to claim 1, wherein the strain is Pseudomonas frederiksbergensis strain of accession number KACC 92246P.
A composition for controlling plant pathogens comprising at least one selected from the group consisting of the strain of claim 1, a culture solution thereof, and a mixture thereof.
The composition for controlling plant pathogens according to claim 8, wherein the plant is ginseng.
A method for controlling plant pathogens, comprising the step of treating the composition for control of claim 8.
11. The method of claim 10, wherein the plant is ginseng (ginseng) plant pathogen control method, characterized in that.

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