KR100942228B1 - Biological control of plant diseases using flavobacterium hercynium epb-c313 - Google Patents

Abstract

PURPOSE: A microorganism formulation using flavobacterium sp. strain is provided to ensure effect of preventing plant disease and prevent residual toxicity and environmental harm. CONSTITUTION: A flavobacterium hercyium EPB-C313(deposit number KACC 91477P) which preventing plant pathogen is isolated from Chinese cabbage. The flavobacterium hercynium EPB-C313 strain well grow in culture medium such as NA(Nutrient Agar), LA(Luria Agar), and TSA(Tryptic Soy Agar) at 15-35°C, pH 5.0-8.5, for 48 to 96 hours. The microorganism formulation for preventing plant pathogen contains flavobacterium hercynium EPB-C313 strain or culture liquid as an active ingredient. The plant pathogen is Plasmodiophora brassicae, Streptomyces acidiscabies or Phytophthora infestans.

Description

Biological control of plant diseases using Flavobacterium hercynium EPB-C313}

The present invention relates to a plant disease control agent using Flavobacterium hercynium EPB-C313 (Microorganism Accession No. KACC91477P) strain isolated from a plant and cultures thereof.

Recently, interest in biological control of plant diseases is increasing. Instead of using chemically harmful pesticides that are harmful to the environment in response to increasing consumer demand for safe agricultural products, biological control selectively uses microorganisms or plant extracts to selectively control the disease without affecting the environment, other plants, and killing.

Therefore, in recent years, a growing number of farmers pursuing eco-friendly agriculture using microbial fungicides have been increasing rapidly, and development of control techniques for producing more efficient and safer agricultural products is required (Dennis and Davis, 1979; Hunter et al., 1987; Katan, 1980; Kim et al., 1995).

Chinese cabbage root-knot disease is a contagious soil infection that is difficult to control. Fungicides used for cabbage root-knot disease are not very effective. In addition, the cost of control is expensive, and because chemical pesticides are used, it exhibits toxicity to shrinkage and causes resistance of drugs and pesticide residues. In particular, the cabbage, cabbage, radish is used for kimjang and raw foods, so the safety problem for pesticides from the consumer has been continuously raised. Therefore, in recent years, cultivated farmers pursuing eco-friendly agriculture using microbial pesticides are increasing rapidly, and development of control technologies for producing safe and safe agricultural products is required.

Chinese cabbage is one of the most cultivated vegetables in Korea, but it is one of the important income crops, but recently, the occurrence of cabbage root-knot disease has increased rapidly, and it is emerging as a problem that needs to be solved urgently (Kim Choong Hoe, 2003; Kim Doo Wook, 1997; Kim Choong Hoe, etc.) . Chinese cabbage root disease is caused by Plasmodiophora brassicae and includes cabbage ( Brassica campestris subsp. Pekinensis ), cabbage ( B. oleracea var. Capitata ) and turnip ( B. campestris var. Rapifera ). It is a disease that occurs worldwide in cabbage and crops (Yoshikawa and Buczacki, 1978). When cabbage develops this disease, underground areas multiply abnormally and above ground growth is inhibited (Arie et al., 1998). If the disease occurs badly, the ground section will wither and lose its merchandise. P. brassicae , a root-knot bacterium, is an absolute parasite belonging to the Protozoa Plasmodiophoromycota, and dormant spores survive in the soil for a long time and become infectious agents (Ariea et al. 1998; Kim Chung Hoe et al., 2000a, 2003).

Until now, the artificial culture technology of root fungus has not been established, so it is not easy to study the distribution and density change of root fungus in soil. In addition, although some chemical pesticides have been developed, effective control methods have not been established, and there is a problem of resistance expression due to various variations of pathogens.

The best way to control the cabbage root gall disease is to block the infectious agents.However, the methods of controlling the already occurring pavement are the seedling methods such as the removal of diseased roots, soil acidity correction, crop rotation, and the cultivation of resistant varieties and heteroconium chattospira ( Heteroconium chaetospira ), physical methods such as biological methods using biocides, chemical methods using fungicides, and heat treatment of soil using solar heat have been proposed (Horiuchi et al. 1982; Tanaka, 1996).

As a biological control, a biological control study using endophytic bacterium, which is advantageous in proliferation in plants, is under progress, especially under conditions in which crop growth conditions are deteriorated. Plant endogenous bacteria are colonized on plant tissues, including seeds and seedlings of plants, fruits, stems, roots, and tubers, but do not cause disease. Plant endogenous bacteria are onions and potatoes (Frommel et al., 1991). Wholesome plants such as corn (Fisher et al., 1992), cotton (Misaghi and Donndelinger, 1990), Kallar grass (Reinhold-Hurek and Hurek, 1998), sugar cane (James and Olivares, 1998) and rice (Barraquio et al., 1997) Separation at has been reported. Endogenous bacteria are mainly colonized in conduit tissues (Frommel et al., 1991; James and Olivares, 1998) or conduits and cortical tissues (Hurek et al., 1994). In rice, the number of endogenous bacteria is 10 ⁵ to 10 ⁸ per g dry weight. It has been confirmed to exist (Barraquio et al., 1997).

Endogenous bacteria have been reported to be involved in plant resistance as well as fungal inhibition (Press et al., 1997). Endogenous bacteria that antagonize have an ecological status similar to that of pathogens, but have an advantage in controlling pathogens compared to control using soil or rhizosphere bacteria. The novel Flavobacterium hercynium EPB-C313 strain of the present invention is excellent in the control effect against Chinese cabbage root gall by Plasmodiophora brassicae , a cabbage root gall pathogen. Do. In addition, as a microorganism that can replace the chemical pesticides, it can prevent the agricultural ecosystem problems caused by residual pesticides and can be effectively used for environmentally friendly control.

The present invention is Flavobacterium hercinium EPB-C313 ( Flavvobacterium) having an effect of suppressing the onset against Chinese cabbage root gall hercynium EPB-C313, Microbial Accession No. KACC91477P) strain to provide a microbial agent for the control of various plant pathogens, including Chinese cabbage root-knot disease.

This invention makes the composition and control methods of plant diseases, such as root-knob disease, using Flavobacterium hercinium EPB-C313.

First, isolates of endogenous bacteria from the roots and stems of Chinese cabbage plants, select strains with excellent plant disease suppression effect through bioassay, and then identify strains by comparing morphological, physiological and biochemical characteristics and DNA profiles. It was. As a result, the strain was identified as Flavobacterium hercynium EPB-C313 (Microbial Accession Number KACC91477P).

The present invention provides a culture condition of the Flavobacterium hercinium EPB-C313 strain. For industrial use, a method of culturing the strain in large quantities in a short time is essential. Flavobacterium hercinium EPB-C313 strain of the present invention grows well on NA (Nutrient Agar), LA (Luria Agar), TSA (Tryptic Soy Agar) medium as a culture medium, growth in the temperature range of 15 ~ 35 ℃ Possible, the growth pH range is 5.0 ~ 8.5, the incubation time is good 48 ~ 96 hours, suitably around 72 hours is best.

The present invention provides a plant pathogen control agent using the Flavobacterium hercinium EPB-C313 strain and a method for controlling the plant pathogen using the same.

The target bacteria that can be controlled using the Flavobacterium hercinium EPB-C313 strain of the present invention are Plasmodiophora brassicae , which is a root-knot bacillus of the slime mold, and Phytophthora infestans, which is a late blight of the fungus, Streptomyces acidiscabies Is applicable.

 Flavobacterium hercinium EPB-C313 strain of the present invention can be controlled by inhibiting the onset by the plant pathogen.

As described in detail above, the microbial preparation using the novel Flavobacterium hercynium EPB-C313 (Microorganism Accession No. KACC91477P) strain according to the present invention effectively prevents various plant pathogens including cabbage root-knot disease. I could control it.

In addition, by enabling the production of safe agricultural products, it is possible to reduce the environmental pollution by increasing the income of farmers, raising the issue of pesticides of consumers, and reducing or replacing the use of chemical pesticides.

Therefore, the present invention can function as a biopesticide against various kinds of Chinese cabbage and crop root root disease, and can prevent problems caused by pesticide residue, and provide a breakthrough biological control method for the preservation of agricultural ecosystem and comprehensive control of the disease. It works.

Hereinafter will be described in detail with reference to a preferred embodiment of the microbial agent and method for the control of root gall disease using Flavobacterium hercinium EPB-C313 ( Flavvobacterium hercynium EPB-C313, microbial accession number KACC91477P) according to the present invention. do. The following examples are merely intended to specifically illustrate the present invention, the plant disease control range of the present invention is not limited to the following examples.

Example 1 Isolation and Antibacterial Activity of Antagonists

The antagonist microorganisms of the present invention are endophytic bacteria, and the roots and stems of Chinese cabbage and crops were sliced, surface sterilized with 70% alcohol for 1 minute, washed with sterile water, placed in PDA medium, and incubated at 27 ° C. for 5 days. . Greenhouse pot test and dilution plate method were performed to test the effect of isolated endogenous bacteria on the control of Chinese cabbage root. The collected endogenous bacteria were shaken in a TSB (Tryptic Soy Broth) medium at 150 ° C for 3 days at 150 rpm, and the culture medium was immersed in 30-day-old Chinese cabbage seedlings for 6 hours. 1 x 10 ⁵ spores / ml) were immersed and transplanted into a vinyl pot, and the incidence of endogenous bacteria with antagonistic ability was expressed as an incidence index (0-4). . Dilution plate method shows the antibacterial activity against phytopathogens by replacing the major phytopathogens in NA (Nutrient Agar) medium.

 Treatment contents  Root Humps Index (0 ~ 4)  Inhibition rate of root nodule formation (%)  Susceptible Breeds  Resistance varieties (CR Singh)  Susceptible Breeds  Resistance varieties (CR Singh)  Antagonist microorganisms (endogenous bacteria)  0  0  100  100  No treatment  3.5  1.2  -  -

 Mycelial growth inhibition level  Pathogen  ++ Clubroot (Plasmodiophora brassicae )  ++ Streptomyces acidiscabies )  ++ Plague ( Phytophthora infestans )  Note) +, 10 mm or less; ++, 10.1-20 mm; +++, 20.1-30; ++++, 30.1 mm or more

Table 1 shows the results of measuring the antagonistic force of Flavobacterium hercinium EPB-C313. 1 is a photograph showing the ability to inhibit the formation of cabbage root knot of Flavobacterium hercinium EPB-C313. For reference, Figure 7 is a photograph of the antagonistic power of Flavobacterium hercinium EPB-C313 against the fungus Phytophthora infestans and Figure 8 is a flabobacter for Streptomyces acidiscabies actinomycetes This is a photo of the antagonistic force of Leeum Hersium EPB-C313.

When confirming the results of Table 1, Figure 1, Figure 7 and Figure 8 the antagonist microorganism of the present invention exhibited excellent antagonistic power, such as root gall disease, dermis disease and late blight. The Flavobacterium hersinium EPB-C313 was incubated for about 3 days in NB (Nutrient Broth) for long-term storage, and the culture medium was added to a sterile 20% glycerol tube and mixed well to make a preserved strain. Store in freezer.

Example 2 Characteristics of Antagonist Microflora Bacterium Hercinium EPB-C313

After inoculating the selected antagonist Flavobacterium herbium EPB-C313 into TSA medium and incubating for 3 days at 28 ° C for 3 days, the colony characteristics were observed, and it was circular orange with a double umbonate. (See FIG. 2). The growth temperature of the antagonist Flavobacterium hercinium EPB-C313 strain of the present invention was investigated by using a temperature gradient device (Temperature Gradient Incubator TN2148, Advantec.), The antagonist of the present invention is Flavobacterium hercini The microbial strain EPB-C313 was able to grow in the range of 15 ~ 35 ℃ and grew best at 25 ~ 30 ℃, and the optimum pH was 6.0 ~ 7.0.

<Example 3. 16S ribosomal RNA (rRNA) gene sequencing analysis of antagonist Flavobacterium hercinium EPB-C313>

Identification of antagonist microorganisms was carried out by the size and shape of the morphology, and the physiological investigation was carried out by temperature response, pH and various biochemical experiments. Molecular biological identification was confirmed by phylogenetic location by separating the DNA and following the nucleotide sequence of 16S rRNA. 27F (5′-GTG CTG CAG AGA GTT TGA TCC TGG CTC AG-3 ′) and 1492r (5'-CAC GGA TCC TAC GGG TAC) to amplify 16S rRNA of antagonist Flavobacterium hercinium EPB-C313 CTT GTT ACG ACT T-3 ') primers were used. The base sequence of the 16S rRNA gene was searched for homology with the DNA database of NCBI GeneBank, and the phylogenetic position was confirmed using the CLUSTAL X program and the PHYLIP program. Molecular systematic analysis based on the nucleotide sequence of 16S rRNA (see FIG. 3), Flavobacterium hercynium is a strain belonging to a systematic group including species of the genus Flavobacterium hercynium . (AM177627) and 99% was confirmed to show a flexible relationship (see Fig. 4).

According to the above results, the antagonistic microorganism of the present invention was identified as Flavobacterium hercynium and named as Flavobacterium hercinium EPB-C313 at the Rural Development Administration Korea Agricultural Microorganism Resource Center. Deposited with 20 days microbial accession number KACC91477P.

Example 4 Flavobacterium hersium Control Effect of Root Knock Disease on EPB-C313 Strains>

In order to confirm the control effect of Chinese cabbage root-knot disease using Flabobacterium hercinium EPB-C313 strain and its culture solution, the cabbage seedlings were immersed in the strain culture medium (concentration 1.0 ㅧ 10 ⁸ cells / ml) 5 days before the formulation and endogenous. Soil mixing treatment of bacteria used a carrier in a mixed condition of skim steel (85% by weight) + castor foil (10% by weight) + zeolite (5% by weight) commonly used in the art for settlement in soil.

In order to verify the control effect of the cabbage root gall disease of the endogenous bacterium Flavobacterium herbium EPB-C313 in the cabbage, the cabbage seedlings were soaked in the endogenous bacterial culture for 6 hours in a plastic house of 660㎡ in which root gall disease occurred. The cabbage was grown in vivo for 5 days. In addition, after centrifugation of the culture medium (10 ⁸ cfu / ㎖) cultured in a titration medium for 3 days (10 ⁸ cfu / ㎖) to recover only the cells, and diluted to a concentration of 10 ⁸ cfu / ㎖ in sterile water carrier (85 weight percent degassing, 10% by weight castor foil, 5% by weight zeolite) was mixed with soil and cabbage seedlings were formulated to determine the control effect. The survey method was the root incidence index (0 ~ 4).

 Incidence Index  Foot disease  0   Without lumps  One   Sometimes small bumps form in the muscle  2   Occasional small bumps on the main and lateral muscles  3   Many small lumps are formed in the main and the long muscles  4   Many large lumps are formed in the main and the long muscles

In addition, the disease control effect is shown in Table 4 as the rate of root-knock formation inhibition and each experiment was repeated three times. FIG. 5 is a photograph showing that the cabbage is cultivated with Flavobacterium hercinium EPB-C313, and FIG. 6 is a photograph showing that nothing is processed when the cabbage is grown.

 Treatment contents  Root Nose Disease Index  Root gall formation inhibition rate (%) Ⅰ (Repeat times) Ⅱ (Repeat times) Ⅲ (repeat count)  Average      EPB-C313 Dipping seedlings 0.56 0.40 0.28 0.41 82.4      EPB-C313 Soil Admixture 0.63 0.36 0.17 0.39 83.3         No treatment 2.60 2.50 1.90 2.33 -

The results of Table 4, Figure 5 and Figure 6, the endogenous bacterium Flavobacterium hercinium EPB-C313 strain significantly reduced the root nodule formation rate when seedling immersion or soil admixture before cabbage formulation was 82.4 ~ 83.3 % Control effect was shown. Simultaneously performing these two methods and one-time soil irrigation of the Flavobacterium hersium EPB-C313 strain culture at the early stage of development during the growth period will increase the control effect. In addition, when the antagonist Flavobacterium hercinium EPB-C313 of the present invention is commercialized, it can be widely used in farms as an effective microbial control agent that can control a wide range of diseases.

Figure 1 is treated with the cabbage of the plabobacterium hercinium EPB-C313 strain (left) and not (right) to the root-knot pathogen Plasmodiophora brassicae ( Psmodiophora brassicae ) in each Photograph showing the results of inhibition (right) or occurrence (right) of root gall formation.

Figure 2 is a colony photograph when the cultured Flavobacterium hercinium EPB-C313 strain in TSA (Tryptic Soy Agar) medium.

3 is a nucleotide sequence of the 16S rRNA gene of Flavobacterium hercinium EPB-C313 strain.

Figure 4 is a format diagram showing the 16S rRNA gene phylogenetic location of the Flavobacterium hercinium EPB-C313 strain.

Figure 5 is a photograph showing the inhibition of Chinese cabbage root-knot disease when treated with Flavobacterium hercinium EPB-C313 strain.

Figure 6 is a photograph showing that the cabbage beak disease was not generated because the treatment with Flavobacterium hercinium EPB-C313 strain.

Figure 7 is a photograph showing the inhibition of the growth of the pathogen when incubated with the Plabobacterium hercinium EPB-C313 strain and the fungus phytophthora infestans .

FIG. 8 is a photograph showing the inhibition of the growth of pathogens when the cultured Plabobacterium hercinium EPB-C313 strain and actinomycetes Streptomyces acidiscabies are replaced.

<110> Chungcheongnam-do <120> Biological control of plant diseases using Flavobacterium          hercynium EPB-C313 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1077 <212> DNA <213> Flavobacterium hercynium EPB-C313 <400> 1 gggtgcgtaa cgcgtatgca atctaccttt tacagaggga tagcccagag aaatttggat 60 taatacctca tagtatacag actcggcatc gagattgtat taaagtcaca acggtaaaag 120 atgagcatgc gtcccattag ctagttggta aggtaacggc ttaccaaggc tacgatgggt 180 aggggtcctg agagggagat cccccacact ggtactgaga cacggaccag actcctacgg 240 gaggcagcag tgaggaatat tggacaatgg gcgcaagcct gatccagcca tgccgcgtgc 300 aggatgacgg tcctatggat tgtaaactgc ttttatacga gaagaaacac tgcttcgtga 360 agcagcttga cggtatcgta agaataagga tcggctaact ccgtgccagc agccgcggta 420 atacggagga tccaagcgtt atccggaatc attgggttta aagggtccgt aggcggttta 480 ataagtcagt ggtgaaagcc catcgctcaa cggtggaacg gccattgata ctgttaaact 540 tgaattatta ggaagtaact agaatatgta gtgtagcggt gaaatgctta gagattacat 600 ggaataccaa ttgcgaaggc aggttactac taatggattg acgctgatgg acgaaagcgt 660 gggtagcgaa caggattaga taccctggta gtccacgccg taaacgatgg atactagctg 720 ttggaagcaa tttcagtggc taagcgaaag tgataagtat cccacctggg gagtacgttc 780 gcaagaatga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 840 aattcgatga tacgcgagga accttaccaa ggcttaaatg tagtttgacc gatttggaaa 900 cagatctttc gcaagacaaa ttacaaggtg ctgcatggtt gtcgtcagct cgtgccgtga 960 ggtgtcagtt aagtcctata cgagcgcaac ccctgtgtag ttgcagcgag tcatgtcggg 1020 aactctacag actgcngtgc aaactgtgag aaggtgggga tgacgtcaat catcacg 1077  

Claims (5)

Flavobacterium hercinium EPB-C313 ( flavobacterium hercynium) that can control plant pathogens EPB-C313, Microbial Accession No. KACC91477P). Microbial preparations for the control of plant pathogens containing the cells of Flavobacterium hersinium EPB-C313 strain or its culture as an active ingredient. The method of claim 2, The plant pathogen is a microbial agent, characterized in that selected from Plasmodiophora brassicae, Streptomyces acidiscabies, Phytophthora infestans . A method for controlling plant pathogens by treating a plant of claim 1 or the microbial agent of claim 2. The method of claim 4, wherein The plant pathogen is a method for controlling plant pathogens, characterized in that selected from Plasmodiophora brassicae, Streptomyces acidiscabies, Phytophthora infestans .

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