KR20100085761A - A novel arthrobacter sp. and biopesticide comprising the strain against tomato grey mould - Google Patents

Abstract

PURPOSE: A novel Arthrobacter sp. strain and an agricultural microorganism formulation containing the same are provided to ensure antibacterial activity and to prevent tomato gray mold. CONSTITUTION: An Arthrobacter oxidans strain has an effect of preventing Botrytis cinerea. The strain is Arthrobacter sp. Ahn005(KACC91402). A composition for preventing or treating tomato gray mold contains the Arthrobacter sp. Ahn005(KACC91402) and agriculturally acceptable carrier.

Description

A novel strain belonging to the novel Atrobacter and a microbial agent for controlling tomato gray mold, including the strain [A novel Arthrobacter sp. and biopesticide comprising the strain against tomato gray mold}

The present invention relates to a novel microorganism belonging to the genus Arthrobacter sp. ( Arthrobacter sp.) And an agricultural microbial agent comprising the strain, and more particularly, tomato lye mildew disease, which has recently caused serious damage in tomato cultivation complexes. In order to develop an antimicrobial substance against the present invention, the present invention is to investigate the antimicrobial substance from Arthrobacter sp.

In general, tomato gray mold disease is called gray mold disease, gray rice disease, gray mold rot disease and is caused by Botrytis cinerea fungus. This disease is one of tomato's main plant diseases, and it occurs mainly in winter plant cultivation rather than road cultivation. Aseptic mildew disease occurs when low temperature around 20 ℃ and humid environment persist, pathogens contaminated with pathogens, fragments of organic matter or soil form grape-shaped conidia and spread to surrounding areas. .

Tomato gray mold disease occurs during the entire growing season, in the first half of the growth, spots with brown or dark brown needles on the leaves or stems are formed. In the late stage of growth, it occurs in all parts including fruit, especially in the navel area of fruit, causing a lot of damage, and rapidly decaying with yellowish white spots of about 1-2mm. (George N. Agrios, Plant pathology, World Science, 5th edition, p. 556, 2006)

In order to control tomato gray mold disease, there is a physical control method that removes infected plants or lowers humidity through artificial temperature rise and ventilation, but the effect of controlling gray mold disease is not great. Therefore, it mainly depends on chemical control, which is sprayed alternately with strong fungicides such as peckhexamide, fludioxonil, benomil and diesofencarb combination, diclofloanid and procmidone. Assay fungal pathogens are easily generated pathogens that are resistant to genetic mutations at the point of action, and the resistant bacterium density increases rapidly, so it is necessary to circulate the fungicide to maintain control effects. (Ziogas BN et al, 2005, Effect of phenylpyrrole-resistance mutations on ecological fitness of Botrytis cinerea and their genetical basis in Ustilagomaydis.European J. of Plant Pathology, 113, 83-100) Therefore, infection occurs during the storage and sale period of infected fruits, which is more urgently necessary to control the harvest before harvesting, and the application of pesticides is more limited due to the stricter standards of residual pesticides. The same various control methods are further required. (Kalogiannis S. et at, Selection and evaluation of phyllosphere yeast as biocontrol agents against gray mold of tomato, 2006, 116: 69-76)

Therefore, environmentally friendly control methods using safer biological control agents to reduce the risks of producers due to misuse of pesticides, consumer safety problems due to pesticide residues after tomato harvest, increase resistance to excessive pesticide use, and to suppress bio-environmental side effects. This is necessary.

The present invention solves the above problems and the object of the present invention to be devised by the necessity of the above is to provide a strain that exhibits a control effect against tomato gray mold.

Yet another object of the present invention is to provide a biochemical pesticide composition comprising the above strain.

Another object of the present invention is to provide a method for controlling tomato ash fungus.

In order to achieve the above object, the present invention provides an Atobacter oxydans strain having a control effect against tomato gray mold.

In a preferred embodiment of the present invention, the strain is preferably an Atobacter Ahn005 strain (KACC91402), but is not limited thereto.

In one embodiment of the present invention, the strain is preferably one having a control activity against Botrytis cinerea , but is not limited thereto.

The present invention also provides a composition for the prevention or treatment of tomato gray fungal infection, comprising the strain of the present invention and an agrochemically acceptable carrier.

The present invention also provides a method for preventing or treating infection of tomato ash fungus by treating the strain of the present invention or a culture thereof or the above composition of the present invention on plant parts or soil.

In the present invention, a microorganism having a control effect on tomato ash fungus is a microorganism having the ability to prevent the occurrence of tomato ash fungus or to cure a plant disease by treating the plant with the microbe, such as spraying the microbe on the plant. Say

The microorganism used for this invention can be used individually by 1 type, or can simultaneously use other multiple types which have the same effect.

The method of culturing the microorganism used for this invention is not specifically limited, It can be set as a regular method. For example, microorganisms may be cultured in a proliferative medium, and the cells may be recovered by means of centrifugation or the like.

The microorganisms used in the present invention are live microorganisms. In addition, although the live microbe of the microorganism used for this invention may not be a spore, it is preferable that it is a spore from a viewpoint of the preservation | save as a product of a microorganism pesticide composition. Therefore, in order to form spores, it is desirable to prepare so that the culture conditions such as the composition of the medium, the pH of the medium, the culture temperature, the culture humidity, the oxygen concentration at the time of culturing and the like are suitable for the spore forming conditions at the end of the culture. Do. In the present invention, when using spores of microorganisms, it is preferable to set the water content of spores to 20% by weight or less from the viewpoint of preservation of the microorganism pesticide composition.

In one embodiment of the present invention, the present invention provides a composition for the prevention and treatment of tomato gray fungal disease containing an effective amount of the genus Atobacter strain and comprising an agrochemically acceptable carrier. The composition of the present invention is formulated as a powder, pellet or granules, microcapsules, etc. according to a microbial pesticide preparation method of a method known in the art, such as to include culture of cultured cells, lyophilized cells and starch, crude protein and rock powder, etc. It can be used as it is. The compositions are surface activators, inert carriers, preservatives, wetting agents, feed promoters, attractants, encapsulating agents, binders, emulsifiers, dyes, U.V. It can be obtained by adding other components which are easy to apply depending on the target pathogen and crops, such as a protective agent and a buffer. The composition of the present invention may be in a form or concentrate or primary composition suitable for direct application by dilution with an appropriate amount of water or other diluent prior to application. It will be apparent to those skilled in the art that the nematicidal concentration may vary, particularly with concentration or direct use, depending on the nature of the particular formulation, the type of plant applied, the soil and climate of the growing region, and the like.

The amount of microorganisms contained in the microbial pesticide composition of the present invention is not particularly limited as long as the effects of the present invention are not impaired, but is 5 to 50% by weight (dry weight) based on the total amount of microorganisms and inorganic salts. ), More preferably 10 to 30% by weight. In addition, the concentration of microorganisms contained in the microbial pesticide composition of the present invention is the colony forming unit, preferably 1 × 10 ⁵ to 1 × 10 ¹⁵ cfu / g, more preferably 1 × 10 ⁹ to 1 × 10 ¹² cfu / g.

There is no restriction | limiting in particular as an inorganic salt used for this invention unless it interferes with the effect of this invention, For example, carbonate, sulfate, phosphate, nitrate, etc. are mentioned, Carbonate and sulfate are preferable, As sodium carbonate, sodium hydrogencarbonate is mentioned. Moreover, sodium sulfate is especially preferable as a sulfate.

The amount of the inorganic salt contained in the microbial pesticide composition of the present invention is not particularly limited as long as the effect of the present invention is not impaired. However, the amount of the inorganic salt is preferably 10 to 50% by weight based on the total amount of the microorganism, the inorganic salt, and the like. It is more preferable to set it as weight%. Incorporation of inorganic salts in these ranges results in a well-balanced microbial pesticide composition in view of the control effect against plant diseases, the hydration of the microbial pesticide composition, and the reduction of roughness of the crop surface caused by the spraying of the microbial pesticide composition. Because.

In addition, the microbial pesticide composition of the present invention may be included even if it does not contain a surfactant, but more preferably included. It is because dispersibility and suspension property in the sparging liquid of the microorganism which is an active ingredient improve when a microbial pesticide composition is diluted and prepared when a surfactant is contained when surfactant is contained. The surfactant used in the present invention is not particularly limited as long as it does not interfere with the effects of the present invention, and may be any type of surfactant such as anionic or cationic, but anionic surfactants are preferred.

The amount of the surfactant contained in the microbial pesticide composition of the present invention is not particularly limited as long as the effects of the present invention are not impaired. However, the amount of the surfactant is preferably 5 to 30% by weight based on the total amount of the microbial pesticide composition, and is preferably 8 to 20% by weight. It is more preferable to do. This is because when the surfactant is contained in these ranges, the hydration and suspension properties of the microorganisms are improved, the preservation of the microorganism pesticide composition is sufficiently secured, and the damage to plants due to the spraying of the microorganism pesticide composition hardly occurs. .

Further, the microbial pesticide composition of the present invention may contain, in addition to the above-mentioned substances, as long as it does not interfere with the effects of the present invention, such as a water-soluble substance.

There is no restriction | limiting in particular as a formulation of the microorganism pesticide composition of this invention, Although granular form, powder form, liquid phase, etc. may be sufficient, granular form and powder form are preferable. It is preferable that the average particle diameter in that case exists in the range of 1 micrometer-100 micrometers, and it is more preferable to exist in the range which is 1 micrometer-50 micrometers. If the average particle diameter is within such a range, the microbial pesticide composition can be easily handled, and if the particle diameter is 1 μm or less, the spore diameter of the microorganism may be lowered, so that the microorganisms die and control against plant diseases. This is because the effect may not be sufficiently exhibited.

There is no restriction | limiting in particular about the usage method of the microorganism pesticide composition of this invention, For example, 1000 times, 2000 times, 4000 times, etc., dilution with water so that it may become a predetermined dilution ratio determined at the time of pesticide registration, the spraying liquid is produced, and the spraying liquid Using a power sprayer, etc.

It can use by spray-processing in mist shape over the whole plant.

Preferably, the strain of the present invention is Arthrobacter sp. (Athrobacter sp.) Ahn005 KACC91402, and the Agrobacter genus in Korea Agricultural Biotechnology Park (Ok, Seogwi-dong, Suwon-si, Gyeonggi-do, Korea) 441-707) ( Arthrobacter sp.) Ahn005 was deposited under the strain name and accession number KACC91402.

The invention is briefly described below.

The present invention firstly provides a novel Arthrobacter sp. Ahn005 KACC91402 bacteria,

Second, Arthrobacter sp.Ahn005 KACC91402 bacterium provides 16A rRNA gene nucleotide sequence structure, which has the following unique structure, and provides Arthrobacter sp. Ahn005 KACC91402 bacterium, -Homologousity of total base sequence: 98% homology with Arthrobacter oxydans .

Third, to provide bakteo in (Arthrobacter sp.) The culture method of Ahn005 KACC91402 bacteria in art,

Fourthly, there is provided an agricultural microbial agent characterized by the treatment of the culture solution for the control of tomato gray mold.

The present invention shows that a novel culture of Arthrobacter sp. Ahn005 KACC91402 can be used as an agricultural microorganism, which is due to overuse that may appear in controlling tomato ash fungus in conventional chemical pesticides. It has the effect of preventing the destruction of the ecosystem.

Hereinafter, the present invention will be described in detail through non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, the scope of the present invention is not limited by the following examples.

Example 1: A novel Arobacter genus ( Arthrobacter sp.) Selection of selection medium for culturing Ahn005 KACC91402

The selected strain Ahn005 was used as a nutrient broth medium used for culturing Arthrobacter sp. For suspension culture. The medium is composed of beep extract (Beef extract) 3 g / L, Peptone (Peptone) 5 g / L, the incubation temperature is 28 ℃, pH 6.8 and incubated for 10 days.

Example 2: A novel Arobacter genus ( Arthrobacter sp.) Morphological observation of Ahn005 KACC91402

The purely isolated Arthrobacter sp. Ahn005 KACC91402 strain was cultured in nutrient broth (Nutrient broth) medium and observed with a scanning electron microscope (FIG. 1).

Example 3: A novel Arobacter genus ( Arthrobacter sp.) Analysis of 16S rRNA gene base sequence of Ahn005 KACC91402

Genetic analysis of novel Arthrobacter sp. Ahn005 KACC91402 was performed, and sequencing of the 16S rDNA gene was performed as follows. Suspension culture in LB medium for 2 days for DNA extraction and the cells were separated by centrifugation. DNA extraction was performed using Genomic DNA Extraction Kit (Intron). PCR was performed with 30 ng genomic DNA, 0.25 uM 16S rDNA gene primer (fD1 and rP2) in reaction solution (10 mM Tris, pH 8.3; 50 mM KCl; 1.5 mM MgCl ₂ ; 0.2 mM dNTPs; 1 unit Taq DNA polymerase). Amplified using. The amplified fragments were about 1500 base-pairs in size. After PCR reaction (initial 94 ℃ 5 minutes once; 94 ℃ 1 minutes, 58 ℃ 1 minutes, 72 ℃ 2 minutes, 35 cycles, 72 ℃ 10 minutes once) and amplified 16S rDNA gene fragment pGEM-T Easy vector After ligation to (Promega), E. coli DH5α was transformed. Transformed E. coli was provoked in a selection medium (1.5% agar, 50mg / L ampicillin, 100uM IPTG, 50mg / L X-gal) and incubated at 37 ℃ for 12 hours. Identification of the vector containing the amplified 16S rDNA gene fragment was first selected by selecting only the white colony of the blue / white colony, it was inoculated in LB medium and cultured. The culture was centrifuged for plasmid separation and Alkaline lysis was used. The isolated plasmid was digested with restriction enzyme E coRI and confirmed the insertion of amplified 16S rDNA fragment by electrophoresis. The amplified 16S rDNA nucleotide sequence was analyzed by sequencing primer (Table 1) and Big dye terminator v.3.0 (Applied Biosystems) using a sequencing device (ABI3100) after direct sequencing reaction. , Homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI) (SEQ ID NO: 1: Figure 2). As a result, it showed 98% homology with Arthrobacter oxydans .

Primer pair Base standing A 5'-AGAGTTTGATCCTGGCTCAG-3 '(SEQ ID NO: 2) 5'-GACGGGCRGTGWGTRCA-3 '(SEQ ID NO: 3)

Example 4: Atobacter genus Arthrobacter sp.) Control of tomato gray mold by Ahn005 KACC91402 culture medium

The varieties of tomato ( Lycopersicon esculentum Mill.) Were tested using Seogwang tomato purchased from Heung Agricultural Co., Ltd., and the tomato gray fungal pathogen was Botrytis cinerea Pers ex Fr. Tomato seeds were planted in horticultural soil (vice farms), grown in greenhouses, and used for experiments after cultivation to tomato seedlings of two to three leaves. Botrytis cinerea Pers ex Fr strain, a pathogen, was inoculated in potato agar medium (potato dextrose agar) and incubated for 7 days in a dark state at a temperature of 20 ° C. Formed.

By spraying a microbial culture of Ahn005 strain on Seedling tomato processing of a medicament, and 1 day after were air dried, added to sterile distilled water to the culture medium and harvesting the spores, and by irradiating the blood cells to step spore concentration under light microscopy 5x10 ⁵ spores per ml for The suspension was made and then sprayed onto the treated tomato seedlings. Tomato seedlings inoculated with the pathogen were developed by wet treatment for 3 days in a 20 ° C. wet room. After investigating the area ratio of the lesion formed on each tomato leaf, the control value was calculated according to the following equation.

[Equation]

Control value (%) = {(Legn area ratio of 1-treated area / Long-area area of untreated area) × 100}

The results of treatment of the culture solution of Arthrobacter sp. Ahn005 KACC91402 (containing microbial intracellular material) cultured by the method described in Example 1 were compared with the untreated (FIG. 3). Generally, tomato gray mold disease caused by Botrytis cinerea Pers ex Fr causes necrosis on tomato leaves.

Referring to Figure 3, in the non-treated group (right), tomato gray mold caused by the inoculated Botrytis cinerea Pers ex Fr caused all of the tomato leaves rot, the treatment of AP27 bacteria (left) of Botrytis cinerea Pers ex Fr of Ahn005 culture Due to the bactericidal effect on You can see that the tomato leaves are healthy and not rotting.

As a result, when treated with the culture medium of the genus Arthrobacter sp. Ahn005 KACC91402, tomato gray mold control effect was 95%. As a control drug, 100ppm of chlorothalonil was used, which showed 100% control value.

Figure 1 shows the results of observing with a scanning electron microscope ( Arthrobacter sp.) Ahn005 strain cultured in Bennet medium (Scanning Electron Microscope).

Figure 2 shows the results of 16S rRNA gene analysis of a novel Arthrobacter sp. Ahn005.

3 is in bakteo in art (Arthrobacter sp.) As photographs showing tomato gray mold disease control efficacy results of the culture process in Ahn005 fungi, by treating the tomatoes a culture solution of bakteo in (Arthrobacter sp.) Ahn005 KACC91402 to art ash Fungal disease is controlled (left) and untreated (right).

<110> Konkuk University Industrial Cooperation Corp. <120> A novel Arthrobacter sp. and biopesticide comprising the strain          against tomato gray mold <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1381 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA <400> 1 agagtttgat cctggctcag gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgacccgg tgcttgcacc ggtgattagt ggcgaacggg tgagtaacac gtgagtaacc 120 tgcccttaac tctgggataa gcctgggaaa ctgggtctaa taccggatat gactcctcat 180 cgcatggtgg ggggtggaaa gcttttttgt ggttttggat ggactcgcgg cctatcagct 240 tgttggtgag gtaatggctc accaaggcga cgacgggtag ccggcctgag agggtgaccg 300 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg 360 cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc ttcgggttgt 420 aaacctcttt cagtagggaa gaagcgaaag tgacggtacc tgcagaagaa gcgccggcta 480 actacgtgcc agcagccgcg gtaatacgta gggcgcaagc gttatccgga attattgggc 540 gtaaagagct cgtaggcggt ttgtcgcgtc tgccgtgaaa gtccggggct caactccgga 600 tctgcggtgg gtacgggcag actagagtga tgtaggggag actggaattc ctggtgtagc 660 ggtgaaatgc gcagatatca ngaggaacac cgatggcgaa agcaggtctc tgggcattaa 720 ctgacgctga agagcgaaag catggggagc gaacaggatt agataccctg gtagtccatg 780 ccgtaaacgt tgggcactag gtgtggggga cattccacgt tttccgcgcc gtagctaacg 840 cattaagtgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg aattgacggg 900 ggcccgcaca agcggcggag catgcggatt aattcgatgc aacgcgaaga accttaccaa 960 ggcttgacat ggactggaaa tacctggaaa caggtgcccc gcttgcggtc ggttcacagg 1020 tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080 caaccctcgt tctatgttgc cagcgcgtta tggcggggac tcataggaga ctgccggggt 1140 caactcggag gaaggtgggg acgacgtcaa atcatcatgc cccttatgtc ttgggcttca 1200 cgcatgctac aatggccggt acaaagggtt gcgatactgt gaggtggagc taatcccaaa 1260 aagccggtct cagttcggat tggggtctgc aactcgaccc catgaagtcg gagtcgctag 1320 taatcgcaga tcagcaacgc tgcggtgaat acgttcccgg gccttgcaca caccgcccgt 1380 c 1381 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agagtttgat cctggctcag 20 <210> 3 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gacgggcrgt gwgtrca 17  

Claims (6)

Atrobacter oxydans strain having a control effect against Botrytis cinerea . The method of claim 1, wherein the strain is Genus Ahn005 strain (KACC91402). The strain according to claim 1 or 2, wherein the strain has a control activity against tomato gray mold. A composition for preventing or treating tomato gray mold fungus infection comprising the strain of claim 1 or 2 and an agrochemically acceptable carrier. A method of preventing or treating an infection of tomato ash fungus by treating the strain of claim 1 or 2 or a culture thereof with a plant part or soil. A method for preventing or treating infection of tomato ash fungus by treating the composition of claim 4 with plant parts or soil.

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