KR102306405B1 - Method for control of gray mold caused by Botrytis cinerea using oxalate-degrading bacteria - Google Patents

Abstract

The present invention relates to a novel strain having oxalic acid decomposition activity, a composition for controlling gray mold disease, and a control method using the same, and more particularly, to a gray mold disease control technique for treating bacteria having oxalic acid decomposition ability on a target crop. According to the present invention, it is possible to prevent infection or translation by decomposing oxalic acid, a pathogenic factor of the gray mold pathogen, so as not to show the pathogenicity of the gray mold pathogen. It has the advantage of being environmentally friendly because it does not cause pollution.

Description

Strain having an oxalic acid decomposition activity, a composition for controlling gray mold disease, and a control method using the same

The present invention relates to a strain having an oxalic acid decomposition activity, a composition for controlling gray mold disease, and a control method using the same, and more particularly, to a gray mold disease control technique for treating bacteria having oxalic acid decomposition ability on a target crop.

In general, the gray mold pathogen Botrytis cinerea ( Botrytis cinerea ) is produced from vegetable crops such as lettuce, cucumber, tomato, strawberry, and pumpkin, to flower crops such as roses and lilies, and to crops such as blueberries and raspberries, which are fruits. It is a fungal microorganism that causes gray mold disease on crops. This gray mold pathogen has a very wide host range, and continuous secondary infection is possible by the scattering of asexual spores, causing enormous damage to crops due to this disease. In addition, since these crops cause gray mold disease during storage or distribution after harvest, it causes a lot of economic damage to disease management after harvest.

This gray mold disease secretes oxalic acid to lower the pH of the invading site to increase the function of cell wall degrading enzymes secreted by pathogens, or acts as a chelator for divalent cations such as calcium ions by oxalic acid to degrade the cell wall. and also causes disease by incapacitating the control mechanism of plants (Manteau et al. 2003; Microbiol. Ecol. 43:359-366).

Although pesticides for controlling such gray mold disease have been developed, it may cause environmental problems such as environmental pollution by chemical pesticides. In addition, spraying of pesticides in facility cultivation can rather promote over-humidification of the greenhouse environment, which in some cases can cause more damage due to infection.

There have been attempts to control using antagonistic microorganisms as one of the eco-friendly methods, but many efforts are needed for practical use.

A method for controlling gray mold disease by Cupriavidus campinensis as an oxalic acid-degrading bacterium for controlling gray mold disease is disclosed (Schoonbeek et al. 2007; MPMI 20:1535-1544). A known technique for applying the oxalate oxidase gene to plants is EP0636181A1. There is a case in which the control effect of Pseudomonas fluorescens PfMDU2 strain capable of degrading oxalic acid was studied (Nagarajkumar et al. 2005; Microbiological Research 160:291-298).

Accordingly, the present inventors surprisingly isolated a bacterium having excellent oxalic acid decomposition ability while studying a method for controlling a disease caused by a specific plant pathogen, particularly Botrytis cinerea , a gray mold fungus using oxalic acid-decomposing bacteria. The present invention was completed by identifying that the occurrence of gray mold disease was suppressed when oxalic acid-decomposing bacteria were treated on crops.

Accordingly, it is an object of the present invention to provide a novel strain capable of controlling gray mold disease in target crops.

Another object of the present invention is to provide a protein capable of controlling gray mold disease in a target crop.

Another object of the present invention is to provide a composition capable of controlling gray mold disease.

Objects according to the present invention in the above are not limited to the above, and other objects of the present invention are clearly defined by those of ordinary skill in the art to which the present invention pertains through the examples and claims to be described later. can be understood

In order to achieve the above object, one embodiment of the present invention is Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or methylobacter provides Solarium jateu Mani (Methylobacterium zatmanii) ODB35 strain (KACC 92231P).

In one embodiment of the present invention provides a PodA ( Pseudomonas oxalate-degrading protein) protein isolated from the strain.

Here, the protein includes an amino acid sequence represented by SEQ ID NO: 5.

In addition, the protein may be encoded by the nucleotide sequence represented by SEQ ID NO: 4.

In another embodiment of the present invention, a vector comprising the nucleotide sequence represented by SEQ ID NO: 4 is provided.

In addition, it provides a culture solution or a culture filtrate of the strain.

In another embodiment of the present invention, it provides a composition for controlling gray mold disease comprising at least one selected from the group consisting of a strain, PodA protein, vector or culture medium or culture filtrate.

Here, the strain has the ability to degrade oxalic acid, or has the effect of reducing the morbidity rate against the spores of the gray mold pathogen.

In addition, in another embodiment of the present invention provides a method for producing a composition for controlling gray mold disease comprising the step of culturing the strain in a medium to which sodium oxalate (sodium oxalate) is added.

In addition, in another embodiment of the present invention, it provides a method for controlling gray mold disease comprising the step of spraying one or more selected from the strain, PodA protein, vector or culture medium or culture filtrate to a target crop or cultivated land.

In another embodiment of the present invention, there is provided a transformant transformed with the vector.

According to the present invention as described above, there is an effect of preventing infection or translation by decomposing oxalic acid, a pathogenic factor of the gray mold pathogen, so as not to show the pathogenicity of the gray mold pathogen.

In addition, according to the present invention, it is possible to achieve eco-friendly control that does not cause environmental pollution by using nature-friendly microorganisms without using chemicals such as disinfectants.

The effects according to the present invention in the above are not limited to the above, and other effects of the present invention can be clearly identified by those of ordinary skill in the art through the examples and claims to be described later. can be understood

Figure 1 shows the absorbance of the oxalic acid-decomposed bacterial culture of the present invention, showing the growth of bacteria.
Figure 2 shows the measurement result of the residual oxalic acid concentration in the oxalic acid-decomposing bacterial culture of the present invention.
3 shows a schematic diagram based on 16s rRNA sequence analysis of oxalic acid-degrading bacteria of the present invention.
Figure 4 shows the morbidity measurement results in Arabidopsis thaliana treated with the oxalic acid-decomposing bacteria of the present invention.
5 shows the protein expression results of the podA gene of the present invention.
6 shows the results of measuring the oxalic acid resolution of the PodA protein of the present invention.

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

The present invention relates to a strain having oxalic acid decomposition activity, a composition for controlling gray mold disease, and a control method using the same. It is about control technology.

While the present inventors were studying a method for controlling diseases caused by specific plant pathogens, particularly gray mold, Botrytis cinerea , using oxalic acid-decomposing bacteria, bacteria having excellent oxalic acid decomposition ability were isolated, and the present invention The Pseudomonas fluorescens ODB5 strain, Pseudomonas abietaniphila ODB36 strain and Methylobacterium zatmanii ODB35 strain isolated from Culture Collection, KACC) on April 20, 2018.

1) Pseudomonas fluorescens ODB5 (KACC 92230P)

2) Methylobacterium zatmanii ODB35 (KACC 92231P)

3) Pseudomonas abietaniphila ODB36 (KACC 92232P)

Also in accordance with the present invention Pseudomonas fluorescein sense (Pseudomonas fluorescens) ODB5 strain (KACC 92230P), also Pseudomonas EBI Tani pillar (Pseudomonas abietaniphila) ODB36 strain (KACC 92232P) or tumefaciens jateu Mani (Methylobacterium zatmanii) ODB35 strain of methyl ( KACC 92231P) has an excellent control effect on gray mold disease among plant diseases, and is a biological pesticide microbial agent that controls and prevents fungal diseases caused by the gray mold bacterium Botrytis cinerea in particular. It can be useful.

According to an embodiment of the present invention, Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or It provides tumefaciens jateu Mani (Methylobacterium zatmanii) ODB35 strain (KACC 92231P) methyl.

In one embodiment of the present invention provides a composition for controlling plant diseases comprising the strain or its culture solution or culture filtrate as an active ingredient. The culture may be any culture produced from a medium under conditions in which the strain can grow normally. However, any additives commonly used for formulation or preservation may be added to the composition for controlling plant diseases. In addition, preferably as plant diseases, yellow blight, yellow blight (yellow patch), Pythium blight, rhizoctonia blight (large patch), dollar spot, anthrax, gray mold disease, late blight And it may be added in an effective amount to the control composition for the control of wilt disease.

According to one embodiment of the present invention may also Pseudomonas fluorescein sense (Pseudomonas fluorescens) ODB5 strain (KACC 92230P), also Tani pillar in Pseudomonas EBI (Pseudomonas abietaniphila) ODB36 strain (KACC 92232P) or methyl tumefaciens jateu Mani (Methylobacterium zatmanii ) One or more strains of ODB35 strain (KACC 92231P) or a natural antagonist of gray mold disease obtained by separation from a culture solution thereof or a culture filtrate thereof is provided.

The chyeon Yanji wherein the material is also Pseudomonas fluorescein sense (Pseudomonas fluorescens) ODB5 strain (KACC 92230P), also Pseudomonas EBI to Tani pillar (Pseudomonas abietaniphila) ODB36 strain (KACC 92232P) or methyl tumefaciens jateu Mani (Methylobacterium zatmanii) ODB35 It can be obtained by removing the present strain from one or more strains among the strains (KACC 92231P) or its culture medium or its culture filtrate, and treating the culture medium from which the strain is removed with an extraction solvent to extract the protein. The extraction solvent may be an alcohol having 1 to 4 carbon atoms, preferably ethanol, but is not limited thereto.

In an embodiment of the present invention, a natural antagonist for inhibiting gray mold disease of the present invention can be obtained by mixing and reacting the culture solution from which the strain has been removed and ethanol, and then precipitating the protein and evaporating the solution to dryness. The natural antagonist isolated in the present invention may be PodA (Pseudomonas oxalate-degrading protein) protein.

In one embodiment of the present invention, the natural antagonist isolated from the strain may be a PodA (Pseudomonas oxalate-degrading protein) protein, wherein the PodA protein is encoded by a gene comprising the nucleotide sequence shown in SEQ ID NO: 4, or SEQ ID NO: It may include the amino acid sequence represented by 5.

In the present invention, a vector using the nucleotide sequence represented by SEQ ID NO: 4 is provided. In more detail, the present invention may provide a recombinant vector comprising the nucleotide sequence represented by SEQ ID NO: 4. The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this are applied to the method disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001). However, it is not limited thereto.

Vectors of the present invention can typically be constructed as vectors for cloning or as vectors for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. Preferably, it can be constructed using a plant as a host.

In the present invention, the vector is a binary vector for transformation capable of permanently expressing a foreign gene in a plant into which a foreign gene is introduced. It is a storage root-specific recombinant expression vector.

The expression vector according to the present invention can be prepared by inserting the promoter of the present invention using an existing vector used for protein expression as a basic backbone and inserting the nucleotide sequence encoding the target protein into the downstream side of the promoter. .

In the example of the present invention, it was prepared using the pET21b vector or the pCAMBIA 1300 vector.

According to one embodiment of the present invention, the present invention provides a transformant transformed by the recombinant vector.

In order to prepare the transformant of the present invention, it may be carried out according to a generally known method, and according to a preferred embodiment, the transformant is a plant or a microorganism. The PodA gene according to the present invention may be introduced into any plant to impart gray mold disease control activity, and is not limited to plants.

Since the PodA gene according to the present invention has gray mold disease control activity, the transformed plant of the present invention may be characterized in that resistance to gray mold disease is improved for wild-type plants. The term 'plant' used in the present invention will be meant to include not only mature plants, but also plant cells, plant tissues, or seeds of plants that can be cultured or cultivated as mature plants.

On the other hand, also in the embodiment of the present invention Pseudomonas fluorescein sense (Pseudomonas fluorescens) ODB5 strain (KACC 92230P), also Tani pillar in Pseudomonas EBI (Pseudomonas abietaniphila) ODB36 strain (KACC 92232P), bacterium methyl Solarium jateu Mani (Methylobacterium zatmanii ) ODB35 strain (KACC 92231P), PodA protein isolated from the strain, the vector, or the culture medium or culture filtrate, which provides a composition for controlling gray mold disease comprising at least one selected from the group consisting of.

The composition for controlling gray mold disease of the present invention may be in the form of a wettable powder or a suspension concentrate. The composition for controlling gray mold according to the present invention may be prepared in a liquid form, and an extender may be added thereto to be used in the form of a powder, or it may be formulated and granulated. However, it is not particularly limited to the formulation. The wettable powder of the present invention as an active ingredient Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or Methylobacterium jat Mani (Methylobacterium zatmanii) ODB35 strain one or more strains or the culture medium thereof (KACC 92231P), white carbon (white carbon), as a wetting agent as an absorbent sodium bis [2-ethylhexyl] sulfosuccinate, sodium league as a dispersant furnace sulfonate and kaolin as an extender, preferably 10% by weight of Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or methyl a tumefaciens jateu Mani (Methylobacterium zatmanii) ODB35 strain (KACC 92231P) at least one strain or culture fluid thereof, 1% by weight of white carbon (white carbon), 1% by weight of the sodium bis [2-ethylhexyl] sulfosuccinate, 1 weight % sodium lignosulfonate and 87 weight % kaolin.

The liquid hydration agent of the present invention as an active ingredient Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or methylobacterium jateu Mani (Methylobacterium zatmanii) ODB35 strain (KACC 92231P) as one or more strain or culture fluid thereof, a wetting agent and a dispersant MBSC (nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, Sodium bis [20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol), an excipient It may consist of isopropanol as an extender and water as an extender. The liquid wettable powder is preferably 50 wt% Pseudomonas fluorescens ODB5 strain (KACC 92230P), Pseudomonas abietaniphila ODB36 strain (KACC 92232P) or Methylobacterium jatmani ( Methylobacterium zatmanii) ODB35 strain (KACC 92231P) one or more strains or each culture solution in the 4% by weight MBSC, 30, but may be made of water% by weight isopropanol and 16% by weight, but is not limited thereto.

On the other hand, in the present invention, the 'effective amount' is an amount sufficient to produce beneficial or desired results, and after uniformly diluting the control composition with water to control gray mold disease, the target crop and Can be sprayed on arable land. When the wettable powder or liquid wettable powder of the present invention is diluted in water, the concentration of the wettable powder or liquid wettable powder ^{is adjusted to 10 5} to 10 ¹⁰ cfu/ml, preferably 10 ⁸ cfu/ml so that the active ingredient can be in a biologically effective range. can, but is not limited thereto.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following examples are only for specifying the contents of the present invention, and the present invention will not be limited thereby.

<Example 1> Isolation of oxalic acid-degrading bacteria and growth in oxalic acid minimal medium

This example was intended to isolate bacteria as the sole carbon source from the surface of plants, such as strawberries and spinach, which contain a large amount of oxalic acid.

Using a sterile cotton swab, rub the surface of the strawberry fruit and the spinach leaf, and then use this again to apply Oxalate minimal agar medium: Na ₂ C ₂ O ₄ 2 g; K ₂ HPO ₄ 2.7 g; NaH ₂ PO ₄ 0.9 g; NH _{Bacteria were isolated by transferring to 4} Cl 0.9 g; MgSO ₄ 7H ₂ O 0.27 g; CaCl ₂ 2H ₂ O 0.009 _{g; FeSO 4} 7H ₂ O 0.0024 g; Agar 15 g). After culturing for 7 days in an incubator at 28°C, the bacterial colonies that had grown were inoculated into a fresh oxalic acid minimum 1,000 medium, and pure separation was performed. 3 days after inoculating the purely isolated bacteria in a liquid incubator containing 0.2% sodium oxalate in 1/10 TSB (tryptic soy broth), the growth of bacteria was investigated using _{absorbance (optical density; OD 600 ), and the} The results are shown in FIG. 1 .

<Example 2> oxalic acid resolution assay

The oxalic acid degradation assay of oxalic acid-degrading bacteria was performed using a generally widely used oxalic acid assay (Hodgkinson & Williams, 1972; Clinica Chimica Acta 36:127-132) and Biovision's oxalic acid staining assay kit (K663-100 Oxalate). Colorimetric Assay Kit) was used.

After 3 days of inoculation in a liquid incubator with 0.2% sodium oxalate added to 1/10 TSB (tryptic soy broth), the culture medium was centrifuged at 13,000 rpm for 5 minutes using a centrifuge, and the upper layer was placed in a 96 well plate. 50 μl of the solution was transferred.

2 μl of the Oxalate Converter included in the kit was put into a 96-well plate, mixed, and reacted in a 37°C incubator for 1 hour. After 1 hour, the reaction mixture (46 μl of Oxalate Development Buffer included in the kit; 2 μl of Oxalate Enzyme mix; 2 μl of Oxalate Probe) was added and mixed well, followed by reaction in a 37°C incubator for 1 hour. After 1 hour, absorbance (optical density; OD ₄₅₀ ) was measured. Using the measured absorbance, the concentration of oxalic acid remaining after the reaction was converted using the following equation.

Figure 2 shows the measurement result of the residual oxalic acid concentration in the oxalic acid-decomposing bacterial culture of the present invention.

As shown in Figure 2, when the resolution was measured through the oxalic acid concentration value converted through the above formula, the remaining samples of the ODB5 strain isolated from the spinach surface and the ODB29, ODB31, ODB35, ODB36 strains isolated from the strawberry surface were The concentration of oxalic acid was the lowest, and thus, it was determined that the strain was excellent in oxalic acid decomposition ability.

<Example 3> Identification and molecular statistical analysis of oxalic acid-degrading bacteria

Five strains ODB5, ODB29, ODB31, ODB35 and ODB36 with excellent oxalic acid decomposition ability were selected among microorganisms isolated from the surface of strawberries and spinach.

The selected strain was subjected to 16S rDNA sequencing for molecular statistical analysis, and the results are shown as SEQ ID NOs: 1, 2 and 3, respectively. Corresponding strains were identified through NCBI BLAST analysis. Each of the ODB5 strains was Pseudomonas fluorescens , the ODB29, ODB31, and ODB36 strains were Pseudomonas abietaniphila , and the ODB35 strain was methylated. It was found to have a tumefaciens jateu Mani 16S rDNA base sequence with high homology (99%) of (Methylobacterium zatmanii). The molecular statistical relationship analysis results of the 16s rDNA nucleotide sequence are shown in FIG. 3 .

3 shows a schematic diagram based on 16s rRNA sequence analysis of oxalic acid-degrading bacteria of the present invention.

As shown in FIG. 3, from the selected strains, the ODB5 strain belongs to the same group as Pseudomonas fluorescens , and the ODB29, ODB31, and ODB36 strains are Pseudomonas abietaniphila , and ODB35 strain is identified as belonging to the same group as tumefaciens jateu Mani (Methylobacterium zatmanii) methyl.

In the present invention, the ODB5 strain selected above was named Pseudomonas fluorescens , ODB29, ODB31, ODB36 strain Pseudomonas abietaniphila ( Pseudomonas abietaniphila ), and ODB35 strain Methylobacterium jatmani It was named (Methylobacterium zatmanii).

<Example 4> Inhibitory effect of gray mold disease of oxalic acid-decomposing bacteria

The general method used by Schoonbeek et al. was used for the inhibition of gray mold disease (MPMI 20:1535-1544). The ODB5, ODB35, ODB36 strains, which were shown to have excellent oxalic acid decomposition ability in Example 3, were cultured for 3 days in King's B (KB) medium, which is commonly used for general Pseudomonas culture, and diluted in sterile water using absorbance to obtain a bacterial suspension (OD). ₆₀₀ = 0.8) was prepared.

3 days after the bacterial suspension was sprayed on Arabidopsis thaliana, the gray mold pathogen Botrytis cinerea was inoculated with a spore suspension (5×10 ⁵ spores/ml). The morbidity rate at which the disease occurred was calculated through the following formula, and the result is shown in FIG. 4 .

Figure 4 shows the morbidity measurement results in Arabidopsis thaliana treated with the oxalic acid-decomposing bacteria of the present invention.

As shown in FIG. 4 , after 12 days, the transformation by gray mold was confirmed in the non-treated group (water), but the translational progression was not confirmed in the treated group treated with oxalic acid decomposing bacteria.

After 16 days, the occurrence of gray mold disease progressed slowly in the treatment group treated with ODB5 and ODB35 bacteria and was confirmed up to 16%, whereas in the treatment group treated with ODB36 bacteria, the disease was significantly lower than that of the untreated group.

Based on the above results, Pseudomonas abietaniphila ODB36 decomposes oxalic acid, a pathogenic factor of the gray mold fungus, and suppresses the pathogenicity of the gray mold pathogen to prevent infection. It is considered to have useful value as a biological pesticide for controlling gray mold disease using this.

<Example 5> Oxalic acid degradation protein

To be the most effective suppressors Pseudomonas EBI in control of gray mold pathogen Tani pillar (P. abietaniphila) oxalate degradation gene (oxalate decarboxylase) of ODB36 strain was named podA (P seudomonas o xalate- d egrading protein) (nucleotide sequence 4 ). The podA gene was PCR amplified using formylNdeI (5'-GGCCATATGCAAAAGCCTTTGCAAGGG-3') and formylXhoI (5'-GGCCTCGAGAATCAATCCGCGAGCGCGCCA-3') primers, and was cloned into pET21b, a commonly used vector for overexpression of proteins. Protein overexpression was treated with 0.1 mM IPTG and incubated at 28 °C for 3 hours to overexpress protein. The protein obtained after protein overexpression was solubilized by dissolving it in glycine/NaOH (pH8.6), and purified by a Ni-NTA column, and the results are shown in FIG. 5 .

The activity of the purified PodA protein was measured using an oxalate colorimetric assay kit (K663-100 Oxalate Colorimetric Assay kit, Biovision). 6 shows the results of measuring the oxalic acid-degrading enzyme activity of the oxalic acid-degrading protein PodA of the present invention. As shown in FIG. 6 , when the resolution of PodA is measured through the converted oxalic acid concentration, it can be seen that only about 1 mM protein decomposes 75% of oxalic acid. As a result, the PodA protein was found to be useful as a protein biological pesticide for the control of gray mold disease.

<Example 6> podA Gene vector production

The podA gene of P. abietaniphila ODB36, whose ability to degrade oxalic acid, a major pathogenic factor of gray mold disease, was confirmed, was cloned into a vector for plant transformation and transformed. The oxalic acid degradation gene podA of the ODB36 strain was PCR amplified using the primers formylKpnI (5'-GGCGGTACCATGCAAAAAGCCTTTGCAAGGG-3') and fomylBamHI (5'-GGCGGATCCTCAAATCAATCCGCGAGCGCG-3'), and a commonly used plant transformation vector, pCAMBIA 1300 (pCAMBIA 1300). ) was cloned into The vector cloned in this example can be transformed into a major crop to impart gray mold resistance.

As described above, the strain having oxalic acid decomposition activity, the composition for controlling gray mold disease and the control method using the same according to the present invention have been described in detail in specific embodiments of the present invention, but those skilled in the art who understand the spirit of the present invention can use the same Other degenerative inventions or other embodiments included within the scope of the present invention may be easily proposed by adding, changing, deleting, etc. other elements within the scope of the spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the following claims rather than the above detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present invention. should be interpreted as

Agricultural Biotechnology Research Institute KACC92230 20180420 Agricultural Biotechnology Research Institute KACC92231 20180420 Agricultural Biotechnology Research Institute KACC92232 20180420

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Method for control of gray mold caused by Botrytis cinerea using <130> PN1904-194 <150> KR 2018/0060338 <151> 2018-05-28 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 1498 <212> DNA <213> Unknown <220> <223> Pseudomonas fluorescens ODB5 16S rRNA gene <400> 1 agagtttgat catggctcag attgaacgct ggcggcaggc ctaacacat caagtcgagc 60 ggtagagaga agcttgcttc tcttgagagc ggcggacggg tgagtaatgc ctaggaatct 120 gcctggtagt gggggataac gttcggaaac ggacgctaat accgcatacg tcctacggga 180 gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt 240 ggtgaggtaa tggctcacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300 cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360 atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420 cactttaagt tgggaggaag ggcagttacc taatacgtga ttgttttgac gttaccgaca 480 gaataagcac cggctaactc tgtgccagca gccgcggtaa tacagagggt gcaagcgtta 540 atcggaatta ctgggcgtaa agcgcgcgta ggtggttagt taagttggat gtgaaatccc 600 cgggctcaac ctgggaactg cattcaaaac tgactgacta gagtatggta gagggtggtg 660 gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720 accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780 accctggtag tccacgccgt aaacgatgtc aactagccgt tgggagcctt gagctcttag 840 tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 900 aatgaattga cggggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960 aagaacctta ccaggccttg acatccaatg aactttctag agatagattg gtgccttcgg 1020 gaacattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080 gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1140 ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200 acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1260 ggagctaatc ccacaaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320 agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg 1380 tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag tctaaccttc 1440 ggggggacgg ttaccacggt gtgattcatg actggggtga agtcgtaaca aggtagcc 1498 <210> 2 <211> 1446 <212> DNA <213> Unknown <220> <223> 16S rRNA gene Methylobacterium zatmanii ODB35 <400> 2 agagtttgat cctggctcag agcgaacgct ggcggcaggc ttaacacat caagtcgaac 60 gggcacctcc gggtgtcagt ggcagacggg tgagtaacac gtgggaacgt gcccttcggt 120 tcggaataac tcagggaaac ttgagctaat accggatacg cccttttggg gaaaggttta 180 ctgccgaagg atcggcccgc gtctgattag cttgttggtg gggtaacggc ctaccaaggc 240 gacgatcagt agctggtctg agaggatgat cagccacact gggactgaga cacggcccag 300 actcctacgg gaggcagcag tggggaatat tggacaatgg gcgcaagcct gatccagcca 360 tgccgcgtga gtgatgaagg ccttagggtt gtaaagctct tttgtccggg acgataatga 420 cggtaccgga agaataagcc ccggctaact tcgtgccagc agccgcggta atacgaaggg 480 ggctagcgtt gctcggaatc actgggcgta aagggcgcgt aggcggccga ttaagtcggg 540 ggtgaaagcc tgtggctcaa ccacagaatt gccttcgata ctggttggct tgagaccgga 600 agaggacagc ggaactgcga gtgtagaggt gaaattcgta gatattcgca agaacaccag 660 tggcgaaggc ggctgtctgg tccggttctg acgctgaggc gcgaaagcgt ggggagcaaa 720 caggattaga taccctggta gtccacgccg taaacgatga atgccagccg ttggcctgct 780 tgcaggtcag tggcgccgct aacgcattaa gcattccgcc tggggagtac ggtcgcaaga 840 ttaaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 900 aagcaacgcg cagaacctta ccatcccttg acatggcatg ttacctcgag agatcgggga 960 tcctcttcgg aggcgtgcac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020 gttgggttaa gtcccgcaac gagcgcaacc cacgtcctta gttgccatca ttcagttggg 1080 cactctaggg aggctgccgg tgataagccg cgaggaaggt gtggatgacg tcaagtcctc 1140 atggcccttg cgggatgggc tacacacgtg ctacaatggc ggtgacagtg ggacgcgaaa 1200 ccgcgaggtc gagcaaatcc ccaaaaaccg tctcagttcg gattgcactc tgcaactcgg 1260 gtgcatgaag gcggaatcgc tagtaatcgt ggatcagcac gccacggtga atacgttccc 1320 gggccttgta cacaccgccc gtcacaccat gggagttggt cttacccgac ggcgctgcgc 1380 caaccgcaag gaggcaggcg accacggtag ggtcagcgac tggggtgaag tcgtaacaag 1440 gtagcc 1446 <210> 3 <211> 1312 <212> DNA <213> Unknown <220> <223> 16S rRNA gene Pseudomonas abietaniphila ODB36 <400> 3 cctggtagtg ggggacaacg tctcgaaagg gacgctaata ccgcatacgt cctacgggag 60 aaagtggggg atcttcggac ctcacgctat cagatgagcc taggtcggat tagctagttg 120 gtgaggtaat ggctcaccaa ggcgacgatc cgtaactggt ctgagaggat gatcagtcac 180 actggaactg agacacggtc cagactccta cgggaggcag cagtggggaa tattggacaa 240 tgggcgaaag cctgatccag ccatgccgcg tgtgtgaaga aggtcttcgg attgtaaagc 300 actttaagtt gggaggaagg gcattaacct aatacgttag tgttttgacg ttaccgacag 360 aataagcacc ggctaactct gtgccagcag ccgcggtaat acagagggtg caagcgttaa 420 tcggaattac tgggcgtaaa gcgcgcgtag gtggtttgtt aagttggatg tgaaatcccc 480 ggggctcaac ctgggaactg catccaaaac tggcaagcta gagtagggca gagggtggtg 540 gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 600 accacctggg ctcatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 660 accctggtag tccacgccgt aaacgatgtc aactagccgt tgggagtctt gaactcttag 720 tggcgcagct aacgcattaa gttgaccgcc tggggagtac ggccgcaagg ttaaaactca 780 aatgaattga cggggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 840 aagaacctta ccaggccttg acatccaatg aactttccag agatggattg gtgccttcgg 900 gagcattgag acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 960 gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1020 ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1080 acggcctggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1140 ggagctaatc ccacaaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1200 agtcggaatc gctagtaatc gcgaatcaga atgtcgcggt gaatacgttc ccgggccttg 1260 tacacaccgc ccgtcacacc atgggagtgg gttgcaccag aagtagctag tc 1312 <210> 4 <211> 1191 <212> DNA <213> Unknown <220> <223> Pseudomonas abietaniphila ODB36 podA gene <400> 4 atgcaaaagc ctttgcaagg gatccgggtc atcgagctcg ggcaactgat cgccgggccc 60 ttcgccgcca agatactggg tgagtttggg gctgacgtga tcaagatcga gccgccgggc 120 acaggtgatc cactgcgcaa atggcgcctg ctgcatgaag ggacgtccgt gtggtgggcc 180 gtgtcctcac gcaacaagcg ttcggtcacc cttgatttgc gtgaaccgga aggtcaggac 240 atcgtgcgca agctgatcgc cgaagccgac gtgctggtgg agaacttccg tcctggtacg 300 ctcgaaggct ggggcctggg ctggcaagaa ctgcatgcgc tcaatccgaa actggtgatg 360 ctgcgggtgt cagggtacgg ccagaccggt ccataccgtg atcgtccggg tttcggtgtg 420 gtcggtgagg ccatgggcgg tttgcgtcat ctgtcgggcg agccggaccg tacgccggtg 480 cgagtcggcg tttccatcgg cgattcgctg tcggccctgc atggcgtcat tggtgtgctg 540 ctcgcgctcc gccatcgtga ccagaacggt ggcgaaggtc aggaagtcga cgtggcgctg 600 tacgagtcgg tcttcaacat gatggaaagc ctgatccctg aattttcggt gttcggcgcc 660 gtgcgccagc cggcgggcag cagcctgccg ggcatcgcgc cgtccaatgc ctaccgttgc 720 cgcgacgggc gctatgcgct gattgcgggc aacggcgaca gtatctatca gcgtctgatg 780 gccttgatcg aacggcccga tctggccgct gatccggcgc tcgcgcataa cgatggacgg 840 gtgaagcagg ttgagctgat cgacgcggcg atttccgcct gggccgcgag cctggacctc 900 gacgaggtgc tggcaaagct caccgccgag cgaattccgg caggcaagat ctacgacgct 960 gccgacattg ccagtgaccc gcattacaag gcccgcgaca tgctgctgga cagtcgactg 1020 gatgacggca ccccggtcac gctgccgggg atcgtgccca aactgggcag cacgccgggc 1080 ggtgtcagtc gtcctgctcc gacgttgggg cagcacaccg acgaggtgct ggaacagctg 1140 ggtatcgacg ctgggcagcg tgccgaatgg cgcgctcgcg gattgatttg a 1191 <210> 5 <211> 396 <212> PRT <213> Unknown <220> <223> Pseudomonas abietaniphila ODB36 PodA protein <400> 5 Met Gln Lys Pro Leu Gln Gly Ile Arg Val Ile Glu Leu Gly Gln Leu 1 5 10 15 Ile Ala Gly Pro Phe Ala Ala Lys Ile Leu Gly Glu Phe Gly Ala Asp 20 25 30 Val Ile Lys Ile Glu Pro Pro Gly Thr Gly Asp Pro Leu Arg Lys Trp 35 40 45 Arg Leu Leu His Glu Gly Thr Ser Val Trp Trp Ala Val Ser Ser Arg 50 55 60 Asn Lys Arg Ser Val Thr Leu Asp Leu Arg Glu Pro Glu Gly Gln Asp 65 70 75 80 Ile Val Arg Lys Leu Ile Ala Glu Ala Asp Val Leu Val Glu Asn Phe 85 90 95 Arg Pro Gly Thr Leu Glu Gly Trp Gly Leu Gly Trp Gln Glu Leu His 100 105 110 Ala Leu Asn Pro Lys Leu Val Met Leu Arg Val Ser Gly Tyr Gly Gln 115 120 125 Thr Gly Pro Tyr Arg Asp Arg Pro Gly Phe Gly Val Val Gly Glu Ala 130 135 140 Met Gly Gly Leu Arg His Leu Ser Gly Glu Pro Asp Arg Thr Pro Val 145 150 155 160 Arg Val Gly Val Ser Ile Gly Asp Ser Leu Ser Ala Leu His Gly Val 165 170 175 Ile Gly Val Leu Leu Ala Leu Arg His Arg Asp Gln Asn Gly Gly Glu 180 185 190 Gly Gln Glu Val Asp Val Ala Leu Tyr Glu Ser Val Phe Asn Met Met 195 200 205 Glu Ser Leu Ile Pro Glu Phe Ser Val Phe Gly Ala Val Arg Gln Pro 210 215 220 Ala Gly Ser Ser Leu Pro Gly Ile Ala Pro Ser Asn Ala Tyr Arg Cys 225 230 235 240 Arg Asp Gly Arg Tyr Ala Leu Ile Ala Gly Asn Gly Asp Ser Ile Tyr 245 250 255 Gln Arg Leu Met Ala Leu Ile Glu Arg Pro Asp Leu Ala Ala Asp Pro 260 265 270 Ala Leu Ala His Asn Asp Gly Arg Val Lys Gln Val Glu Leu Ile Asp 275 280 285 Ala Ala Ile Ser Ala Trp Ala Ala Ser Leu Asp Leu Asp Glu Val Leu 290 295 300 Ala Lys Leu Thr Ala Glu Arg Ile Pro Ala Gly Lys Ile Tyr Asp Ala 305 310 315 320 Ala Asp Ile Ala Ser Asp Pro His Tyr Lys Ala Arg Asp Met Leu Leu 325 330 335 Asp Ser Arg Leu Asp Asp Gly Thr Pro Val Thr Leu Pro Gly Ile Val 340 345 350 Pro Lys Leu Gly Ser Thr Pro Gly Gly Val Ser Arg Pro Ala Pro Thr 355 360 365 Leu Gly Gln His Thr Asp Glu Val Leu Glu Gln Leu Gly Ile Asp Ala 370 375 380 Gly Gln Arg Ala Glu Trp Arg Ala Arg Gly Leu Ile 385 390 395

Claims (12)

delete Pseudomonas abietaniphila ODB36 strain (KACC 92232P) having gray mold disease control activity. The culture filtrate of the strain of claim 2. delete PodA (Pseudomonas oxalate-degrading protein) protein comprising the amino acid sequence represented by SEQ ID NO: 5 isolated from the strain of claim 2 . PodA (Pseudomonas oxalate-degrading protein) protein that is encoded by the nucleotide sequence represented by SEQ ID NO: 4 isolated from the strain of claim 2 . A vector comprising the nucleotide sequence represented by SEQ ID NO: 4. The culture solution of the strain of claim 2. The strain of claim 2, the PodA protein of claim 5, the PodA protein of claim 6, the vector of claim 7, the culture medium culturing the strain of claim 2, and the culture filtrate of claim 2 comprising at least one selected from the group consisting of , A composition for controlling gray mold disease. delete The strain of claim 2, the PodA protein of claim 5, the PodA protein of claim 6, the vector of claim 7, the culture medium culturing the strain of claim 2, and the culture filtrate of claim 2, at least one selected from the group consisting of a target crop Or gray mold disease control method comprising the step of spraying on cultivated land. A transformant transformed with the vector of claim 7.

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