KR20090105149A - Novel Paenibacillus polymyxa NB1 Having Anti-plant pathogens Activity and Composition Comprising the Same - Google Patents

Abstract

PURPOSE: A novel paenibacillus polymyxa NB1 with anti-bacterial activity and a composition containing the same are provided to prevent plant diseases caused by plant pathogen. CONSTITUTION: A plant pathogen antagonic Paenibacillus polymyxa NB1 has deposit number KFCC 11413P. The plant pathogen is selected from Colletotrichum acutatum, Pythum ultimum, Phytopthora capcisi, Rhizoctonia solani AG4, Botrytis cineriea, and Fusarium oxysporum.

Description

Novel Paenibacillus polymyxa NB1 Having Anti-plant pathogens Activity and Composition Comprising the Same

The present invention relates to a novel F. Bacillus polymyxa NB1 strain having antimicrobial activity against plant pathogens, a composition comprising the same, and a method for controlling plant pathogens by treating the composition.

Plant pathogens cause huge losses to the agricultural economy by infecting plants such as crops and flowers, reducing the yield of the plants and causing deterioration of the quality of products.

Crop diseases caused by phytopathogenic fungi are very limiting factors for stable production of crops along with pests and weeds. Among the various phytopathogenic fungi, many of the fungal nuclei, such as B. cinerea and R. solani, are soil contagious, causing widespread disease in many types of crops. It causes a lot of damage. These pathogens overwinter in the mycelial state and become the primary source of infection in the following year, which can survive in unfavorable environmental conditions in diseased tissues or soil, and can be degraded by spores or mycelium by neighboring antagonist microorganisms. It is also resistant to effective control. Leaf fungus Cradosporium fulvum and Alternaria solani also cause a lot of damage to vegetable crops. In particular, various types of fungicides are currently used for the control of these pathogens, but the development of safe and effective control methods is urgently required as the effectiveness of the drug decreases due to drug resistance due to misuse and abuse of the fungicides.

In order to control such plant pathogens, methods for controlling pathogens include physical methods for improving the cultivation environment, chemical methods using fungicides, and microbiological methods using microorganisms.

However, physical control alone cannot be expected to have a great effect, and chemical control has not only significantly reduced the control effect on the packaging due to the emergence of drug-resistant pathogens, but also leads to serious environmental pollution by spraying a large amount of chemical pesticides. In addition, the use of chemically synthesized pesticides is increasingly regulated due to toxicity to humans and animals, residual toxicity in soil and crops, and environmental pollution due to packaging containers. Its use is greatly limited due to the strengthening of specific regulations on pesticides. Therefore, in recent years, research on microbiological control has been actively conducted as an alternative method, and the development of effective control methods for environmentally friendly agriculture is urgently required.

As an environmentally safe and effective control method, biological control using microbial agents has been conducted by many researchers at home and abroad since the early 1970s. Recently, more than 10 kinds of microbial products have been commercialized in the world, and research on the development of microbial products is rapidly increasing due to the change of social awareness toward environmentally safe sustainable agriculture. Representative microbial agents currently developed and commercialized in foreign countries include Trichoderma harzianum and Trichoderma polysporum, BINAB T (1988, ER ButtsInternational), Glio GlioGard (1990, WR Grace Co.), a bottle control agent for greenhouse plant seedlings using Gliocladium virens, Fstop (1991, Kodak), Trichodex (1997, Israel), using Trichoderma harzianum (Development) and DAGGER, which control seedlings of cotton lipotonia and Pythium sp. (1990, Ecogen Inc.), BLUE CIRCLE (1990, Stine Microbial Products), a seed treatment of several crops using P. cepacia, and antagonistic actinomycetes streptoma Process and the like so Seo irregularities disk (Streptomyces griseoviridis) the Fusarium (Fusarium) wilt, Mo jalrokbyeong control agent MYCOSTOP (1985, Kemira Oy Biocontrol of Finland) using.

In Korea, research on biological control of ginseng root rot has been conducted since the early 1970s, and studies on biological control of red pepper disease, cucumber strawberry wilting disease, sesame seedling disease, and ash fungus have been conducted, but the results are insignificant. In addition, microorganism pesticides are patented microbicides, and bacteria include Bacillus sp. BS555, KCTC 8821P, Bacillus polymoxa B36, FERM BP-6068, and Pseudomonas aureofaciens B5 ), Enterobacter cloacae B51 (FERM BP-6069), and Bom1 (KFCC No. 10992) and Bom2 (KFCC No. 10993) in Streptomyces, actinomycetes that secrete antifungal substances. In addition, as a microorganism having an antagonistic ability against pathogenic fungi Bacillus subtilis var. Amyloliquefaciens KL114, KCTC 8913P, Bacillus amyloliquefaciens KTGB0202 KCTC 10564BP), alkali gin pae Callis (Alcaligenes faecalis KL1179, KCTC 8914P), Pseudomonas aeruginosa KL1121, KCTC 8915P, Pseudomonas aurantiaca KL1326, KCTC 8916P, and Bacillus subtilis and Fluores monolis. A fungal antagonist against Actinomycetes Streptomyces genus includes Pseudomonas fluorescence Bios 1 (Biovar 1), and fungi include Trichodelma hagianium, Penicillium sp., And Aspergillus spp. sp.) and the acute parasitic fungus Acremonium Strictium BCP (Acremonium strictum, KCTC0639BP) A.

The principle of biological control by antagonistic microorganisms is to reduce the density or pathogenesis of pathogens through antibiotics produced by antibiotics, parasitic or phagocytosis by cell wall degrading enzymes, and nutrient competition or niche competition. The main mechanisms of action are cross-protection and induction of host resistance using non-pathogenic or weakly-pathogenic strains. For example, the inhibitory action of fungal diseases caused by antibiotics or volatile substances, toxins, and special inhibitors secreted by the genus Trichoderma and gliocladium is known. On the other hand, antagonism by parasitic or phagocytosis has been actively studied recently. Pathogens caused by chitinase, glucanase, and cellulase, which are cell wall degrading enzymes secreted by antagonistic bacteria Dissolution of is known as the main mechanism of action.

To replace organic synthetic pesticides, we want to identify new microorganisms that can effectively act on plant pathogens.

In this regard, the present inventors selected strains having antimicrobial activity against plant pathogens, and identified strains were novel strains belonging to Fanibacilli polymyxa, and said strains had antimicrobial activity against various kinds of plant pathogens. Reconfirmed.

Hereinafter, the present invention will be described in detail.

In one embodiment, the present invention relates to a novel F. ni. Polymyxa NB1 strain having antimicrobial activity against plant pathogens, deposited with accession number KFCC 11413P.

"Panibacillus polymyxa NB1" of the present invention is a strain isolated from the roots of various plants collected in Hadong, Gyeongnam, morphological, physiological and biochemical characterization and 16V ribosomal rDNA sequence analysis Strains identified as being autologous. The strain of the present invention is broad and excellent growth inhibitory activity, that is, antagonist against various kinds of plant pathogens such as anthrax , phytium ultimum, late blight, Laytononia solani AG4, ash fungus, Fusarium oxysporum It is characterized by showing the action.

 The FB NSA strain was deposited on March 10, 2008 with the accession number KFCC 11413P to the Korean Federation of Culture Colleftions (KFCC) located in Yurim Building, 2F, Hongje-dong, Seodaemun-gu, Seoul.

In another aspect, the present invention relates to a plant pathogen inhibiting composition comprising the F. Panaxylus polymyxa NB1 strain, the culture filtrate, or a mixture thereof.

The culture filtrate is a supernatant from which the cells are removed from the culture solution prepared by culturing and diluting the F. N.B. strain, and can be obtained by centrifugation or filtration.

Panibacillus polymyxa NB1 strain, culture filtrate, or a mixture thereof of the present invention can be usefully used as a plant pathogen inhibitor composition. At this time, the composition of the present invention may include surfactants, plant nutrients, preservatives, bio supplements, surfactants, inorganic salts, adjuvants, binders and extenders commonly used in the art. In addition, the composition of the present invention may include a material that can improve shelf life, for example, pea moss, zeolite, talc and kaolinite, clay, and the like.

The composition of the present invention may be formulated into various formulations such as powders, pellets, granules, and solutions.

The plant pathogen inhibiting composition of the present invention effectively controls various diseases of plants caused by plant pathogens such as anthrax , phytium Ultimum, late blight, lytonia solani AG4, ash fungus, Fusarium oxysporum At the same time, it does not cause problems such as ecosystem destruction, groundwater pollution, residual toxicity of crops, etc.

In another aspect, the present invention relates to a method of inhibiting the growth of plant pathogens by treating a plant pathogen inhibiting composition comprising a Pannibacillus polymyx NB1 strain, a culture medium, or a mixture thereof.

The composition of the present invention can be applied to all plants that are caused or may be caused by the infection of pathogens to which the Penivacillus polymyxa NB1 strain antagonizes. Plant pathogens can be effectively controlled by spraying the composition of the present invention directly on various parts such as seeds, stems and leaves of the plant, or by treating the soil or the like on which the plant is growing. In addition, the composition of the present invention may control pathogens by treating not only the plant being grown, but also plants, such as vegetables, fruits, and flowers, which are stored and distributed after harvesting.

The Fanibacillus polymyxa NB1 of the present invention is anthrax ( colletotrichum acutatum ) , Pythum ( Pythum) ultimum ), phytophthora capsici ), Raiztonia Solani AG4 ( Rhizoctonia solani AG4), Botrytis cineriea and Fusarium oxysporum oxysporum ) shows excellent antibacterial activity against various kinds of plant pathogens.

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

Example  One: NB1  Isolate

After washing various plant roots collected from Hadong, Gyeongnam, with water, crush them using a mortar and pestle, dilute them with sterile water, and dispense and spread them in 10% TS (Triptic Soy Agar) medium. Incubated for 4 days at ℃. A single colony was selected from the cultured medium and re-cultured in TSA medium, and the cultured strain was used as a storage strain.

Example  2: Strain Characterization

In order to investigate the morphological characteristics of NB1 strain, the cell type, size, spores and flagella of NB1 strain were examined by electron microscopy, and colonies growing in TSA medium were examined.

In addition, the Gram staining reaction, growth temperature, growth pH, and acid requirements were investigated for physiological characteristics. Gram staining reaction was investigated using Crystal violet stain solution (Dhingra amp; Sinclair, 1985. Basic Plant Pathology Method. CRC Press). Growth temperature range and optimum growth conditions were determined using TSB (Tryptic Soy Broth) medium. The culture was shaken and cultured at intervals of 5 ° C. under 200 rpm and pH 7 in a range of 15-40 ° C., and the density thereof was investigated using absorbance. Growth pH and optimal growth pH were determined by adjusting the acidity of TSB medium to 3 to 10 pH and shaking culture at 27 ° C. and 200 rpm to determine the density of NB1 strain by measuring absorbance. Oxygen urine composition was determined by inoculating NB1 strain in TSA medium, and then comparing the growth and growth under anaerobic conditions in which nitrogen was injected into airtight containers and general aerobic conditions. As shown in Table 1 below, strain NB1 forms endospores as rod shapes, and has plural flagella. In addition, it is possible to grow at 15 ~ 40 ℃ as gram-positive bacteria, the optimum growth temperature is 25 ~ 30 ℃. The pH range for growth is 4.0 ~ 9.0pH and the optimum growth pH is 6.0 ~ 7.0. Oxygen urine composition measurement results can be seen that aerobic (Aerobic).

Characterization of NB1 Strains of Panibacillus Polymixosa division characteristic Cell type Bacillus Size (μm) 1.2 to 2.3 (thickness), 2.3 to 4.2 (length) Spores Endogenous spore formation flagellum There are plural flagella Gram stain positivity Growth temperature range (℃) 15-40 Optimum growth temperature (℃) 25-30 Growth pH Range 4.0-9.0 Optimal growth pH 6.0-7.0 Oxygen composition Aerobic

Example  3: cell fatty acid analysis

Identification of strains was carried out using the microbial identification method (MIDI Inc., USA) of the strain, using the fatty acid methyl ester according to the operating manual (Version 6.MIDI Inc., USA). After incubation at 27 ℃ for 24 hours, a single colony was taken, extracted through lysis, hybridization, and methylation, and analyzed using gas chromatography. Sim Index) is 0.785Paenibacillus polymyxaNB1 was identified as (Table 2)

Paenibacillus polymyxa Cellular Fatty Acid Assay of NB1 Peak Name Percent C _{14 : 0} ISO 2.87 C _{14 : 0} 3.05 C _{15 : 0} ISO 6.99 C _{15 : 0} ANTEISO 57.42 C _{15 : 0} 1.22 C _{16 : 1} w7c alcohol 0.63 C _{16 : 0} ISO 8.06 C _{16 : 1} w11c 3.54 C _{16 : 0} 7.39 C _{17 : 1} ISO w10c 0.43 C _{17 : 0} ISO 2.65 C _{17 : 0} ANTEISO 5.76

Example  4: Molecular Biological Characterization

To analyze the nucleotide sequence of 16s ribosomal DNA, purely isolated NB1 strain was inoculated in Nutrient broth Agar (Difco) medium, incubated at 28 ° C. for 3 days, and DNA was extracted using Benzyl chloride method. For PCR amplification of 16s rDNA, two oligonucleotide primers 27F (5`-AGAGTTTGATCCTGGCTCAG-3`) and 1492R (5`-GGTTACCTTGTTACGACTT-3`) were denatured at 94 ° C and annealed at 58 ° C. (Annealing), DNA extracted under the conditions of extension (Extension) at 72 ℃ was amplified.

PCR amplification products were analyzed by nucleotide sequence using Cenetic analyzer 310A (Applied Biosystems). The base sequence was searched for homology with the database of DDBJ / NCBI / Genebank.

The analysis shows that NB1 A strain belonging to the phylogenetic group including species of the genus Paenibacillus , Paenibacillus It was identified as representing the relationship of the flexible polymyxa DSM 36t (X57308) and 99%. Thus NB1 is Paenibacillus It was identified as polymxa . The onset of these strains has caused the strain Paenibacillus polymxa Named NB1, it was deposited with the Korea Microbial Conservation Association and received accession number KFCC 11413P.

Example  5: NB1 Control effect

NB1 strain ( Paenibacillus Polymyxa NB1), Rhizoctonia , a Soil Infectious Plant Pathogen for the Growth Inhibition of Phytopathogenic Microorganisms solani AG4, Pythum ultimum , Phytopthora capcisi And Fusarium oxysporum and airborne pathogens, Botrytis cineriea ), anthrax ( Colletotrichum) acutatum ) The growth inhibition effect on phytopathogenic bacteria was investigated by replacement culture. The investigation was carried out on potato agar medium (PDA, Potato Dextrose Agar), in which both phytopathogens and NB1 grow well. After 24 hours of inoculation of the pathogen in the center of the plate, incubate NB1 for 36 hours in TSB medium, soak the paper disk (diameter 8mm) in a fully grown medium for 10 minutes, dry for 1 minute in aseptic state, and place it 3cm away from the pathogen flora. Replaced. This was investigated while incubating for 5-7 days at 28 ℃ as shown in Table 3. The interval between the inhibitory zones formed between the pathogens of the growth inhibitory effect and NB1 was randomly divided into five levels of no, +, ++, +++, and ++++. Got. As shown in Table 3, NB1 has a growth inhibitory effect on all phytopathogens used in the experiment, in particular, Botrytis cineriea , Phytophthora capsici , Colletotrichum Acutatum showed the strongest inhibitory effect on growth.

Paenibacillus Growth Inhibitory Effects of Polymyxa NB1 on Plant Pathogens Plant pathogen Antifungal effect Culture temperature (℃) Survey date Rhizoctonia solani AG4 +++ 28 5 Botrytis cineriea ++++ 28 5 Pythum ultimum ++ 28 5 Phytophthora capsici +++ 28 7 Fusarium oxysporum ++ 28 7 Colletotrichum acutatum ++++ 28 7

Paenibacillus polymyxa NB1 strain of the present invention is a strain having a wide range of antimicrobial activity against a variety of plant pathogens, it is possible to expect a variety of applications in the agricultural field because it is possible to obtain both excellent conservation effect against disease caused by the conservation of agricultural ecosystem and plant pathogens. .

1 is a result showing the antagonistic effect of the plant pathogens of the F. N. Pannibacillus polymyxa, (A) is anthrax (Colletotrichum acutatum), (B) is Pittium Ultimum (Pythum ultimum) (C) is a late blight (Phytophthora capsici), (D) Raiztonia Solani AG4 (Rhizoctonia solani AG4), (E) are gray mold fungi (Botrytis cineriea) And (F) are the Fusarium oxysporum (Fusarium oxysporumAntagonistic effect on).

<110> NAMBO CO LTD <120> Novel Paenibacillus polymyxa NB1 Having Anti-plant pathogens          Activity and Composition Comprising the Same <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 27F primer <400> 1 agagtttgat cctggctcag 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 1492R primer <400> 2 ggttaccttg ttacgactt 19  

Claims (5)

Plant Pathogen Antagonistic Strains Pannibacillus Polymyxa NB1 ( Paenibacillus polymyxa ; Accession number KFCC 11413P). According to claim 1, wherein the plant pathogen is anthrax ( colletotrichum) acutatum ) , Pythum ultimum), reverse germs (Phytopthora capcisi), Rhizoctonia solani AG4 (Rhizoctonia solani AG4), Botrytis cineriea ) and Fusarium oxysporum ( Fusarium oxysporum) , characterized in that at least one strain selected from the group consisting of. A composition for controlling plant pathogens, comprising the F. Pannibacillus polymyxa NB1 strain, a culture filtrate, or a mixture thereof. A method of inhibiting the growth of plant pathogens by treating the composition of claim 3 to a plant or soil. 5. The method of claim 4, wherein the plant pathogen is at least one selected from the group consisting of anthrax , phytium Ultimum, late blight, Laytonia solanie AG4, gray mold and fusarium oxysporum.

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