KR20100085759A - A novel arthrobacter sp. and biopesticide comprising the strain against tomato late blight - Google Patents

Abstract

PURPOSE: A novel strain and a microorganism formulation containing the same are provided to prevent ecosystem damage and potato or tomato late blight. CONSTITUTION: An Arthrobacter oxidans strain has an effect of preventing Phytophthora infestans. The strain is Arthrobacter Ahn004(KACC91401) which is isolated from domestic fermented seafood. A composition for preventing or treating tomato or potato late blight contains Arthrobacter Ahn004(KACC91401) strain and agriculturally acceptable carrier.

Description

A novel strain belonging to the genus Atobacter, and a microbial agent for controlling tomato late blight, including the strain [A novel Arthrobacter sp. and biopesticide comprising the strain against tomato late blight}

The present invention is an invention for controlling tomato late blight, especially as to dispense an antimicrobial substance from bakteo in (Arthrobacter sp.) Fungi by Ahn004 art isolated from domestic Fermented relates to the use of agricultural microbial agent.

In general, not only potatoes, but also tomato late blight are caused by Phytophthora infestans , which occur mainly in the environment where the low temperature of 10 ℃ to 24 ℃ is maintained and the relative humidity maintains high humidity of 90% for more than a week. Gives. (MacKenzie, 1981, Plant disease, 65, 394-399)

Plague caused by phytophthora infestans occurs during the entire growth period of potatoes and tomatoes, mainly invading the foliage and damaging the ground part. Necrosis, which significantly reduces the quality. The pathogen forms an elliptical spore sac, indirectly germinates at 12 ° C to 15 ° C and directly germinates at 15 ° C or higher. Like the name Phytophthora, it causes a lot of damage because it is contagious to surrounding plants. In addition, follicle forms exist in the soil for 3-4 years, resulting in the recombination of genes through wintering and mating, resulting in increased resistance of germs to chemical control and increased incidence density (George N. Agrios, Plant pathology, World Science, 5th edition, p. 422-426, 2006)

Historically, the plague's communicability, damage and impact are well illustrated by the Irish Atmospheric Event, which occurred at the end of the 19th century. The late blight was discovered in 1863 by Anton deBary as an Oomycetes P. infestans . To control potato or tomato late blight, secondary measures such as disease-free seed and disease-resistant varieties are used, but chemical control by disinfectant treatment is mainly used. Currently, metaaxyl and dimethomorph are used as control pesticides, but pathogens are sexually reproduced through cross-breeding, resistant strains and new strains are gradually introduced, and the density of their development is gradually spread. It has been controlled by increasing the use of pesticides or the use of pesticides, which has increased the pathogenic resistance of pathogens and caused environmental problems around the environment.

Therefore, there is an urgent need for the development of new environmentally friendly control methods for the excessive use of pesticides, the suppression of resistant pathogens and the protection of the surrounding ecological environment. In particular, as the interest and consumption of eco-friendly, pesticide-free and low-pesticide agricultural products have increased, the necessity of the development of biopesticides and biochemical pesticides with excellent control of pathogens by using microorganisms is increasing.

The present invention is to solve the above problems and the object of the present invention is to provide a strain having a control activity against potato or tomato late blight in order to solve the above problems.

Another object of the present invention is to provide a new agricultural microbial agent for potato or tomato late blight.

It is yet another object of the present invention to provide a new method for controlling potato or tomato late blight.

In order to achieve the above object, the present invention provides an Atobacter oxydans strain having a control effect against tomato or potato late blight.

In a preferred embodiment of the present invention, the strain is preferred but not limited to Atrobacterus Ahn004 (KACC91401).

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

The present invention also provides a composition for preventing or treating tomato or potato late blight infections 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 or potato late blight by treating the above strains or cultures thereof or the above composition of the present invention to plant parts or soil.

The present invention also provides a method for producing a biochemical pesticide preparation, characterized in that the culturing of the strain, the culturing may be characterized in that carried out for 7-14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium. have.

The present invention also provides a biochemical pesticide preparation comprising the strain or its culture as an active ingredient.

The culture solution may be characterized in that it is incubated for 7-14 days at 25 ~ 30 ℃ in Bennett (Bennet) medium, the biochemical pesticides may be characterized in that for controlling tomato blight.

In the present invention, a microorganism having an effect of controlling tomato or potato late blight is a microorganism having the ability to prevent the occurrence of tomato or potato late blight or to cure a plant disease by treating the plant with the microorganism such as spraying the microorganism 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 or potato late blight, containing an effective amount of the genus Atobacter strain and comprising an agriculturally 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. ), 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, 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, and the spreading liquid is produced, The spreading 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 the bacterium Ahn004 (KACC91401) belonging to the genus Arthrobacter sp., October 7, 2008, Korea Agricultural Biotechnology Parkwon (225, Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do 441-707) Staphylococcus sp . Was deposited under the strain name AP27 and accession number KACC91291P.

The invention is briefly described below.

The present invention firstly provides a bacterium Ahn004 (KACC91401) belonging to the genus Arthrobacter sp.,

Second, the genome sequence of the 16S rRNA of the bacterium Ahn004 (KACC91401) belonging to the genus Arthrobacter sp. (Acc. Sp.) Belongs to the genus Arthrobacter sp. Ahn004 (KACC91401) is provided,-homology of the entire base sequence: 99% homology with Arthrobacter oxydans .

Third, to provide a method for producing a culture medium of the fungus Ahn004 (KACC91401) belonging to the genus Arthrobacter sp.,

Fourth, there is provided a agricultural microbial agent, characterized in that the treatment of the culture solution for the control of tomato late blight.

The present invention shows that the culture medium of the fungus Ahn004 (KACC91401) belonging to the novel Arthrobacter sp. Can be used as an agricultural microbial agent, which is an overuse that can appear in tomato pest control in conventional chemical pesticides. It can prevent the destruction of ecosystem by

Hereinafter, the present invention will be described in detail through non-limiting examples.

However, the following examples merely illustrate the invention and the scope of the present invention is not limited to the following examples.

Example 1 Isolation and Identification of Novel Arobacterium Ahn004 (KACC91401)

Culture medium

Bennet medium used for actinomycetes culture was used for suspension culture of the selected strain KACC91401. The medium was composed of Agar 15 g / L, Beef extract 3 g / L, peptone 5 g / L, the culture temperature was 28 ℃, pH 7.2 and incubated for 10 days.

Example 2: A novel Acrobacter genus ( Arthrobacter morphological observation of the fungus Ahn004 (KACC91401) belonging to

The purely isolated novel Arobacterium Ahn004 strain (KACC91401) was cultured in nutrient broth (Nutrient broth) medium, and observed in a scanning electron microscope (Scanning Electron Microscope) (Fig. 1).

Example 3: A novel Arobacter genus ( Arthrobacter 16S rRNA gene base sequence analysis of Bacillus Ahn004 (KACC91401) belonging to

Genetic analysis of the bacterium Ahn004 (KACC91401) belonging to the genus Arthrobacter sp. 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 by insertion of amplified 16S rDNA fragment by electrophoresis. The amplified 16S rDNA nucleotide sequence was subjected to direct sequencing reaction of PCR fragment using primers (Table 1) and Big dye terminator v.3.0 (Applied Biosystems) prepared by our sequencing apparatus (ABI3100). The homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI) (SEQ ID NO: 1; FIG. 2). As a result, it showed 99% 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 Control of Tomato Blight on Cultures of A. 004 (KACC91401)

The varieties of tomato ( Lycopersicon esculentum Mill.) Were tested by Seokwang tomato purchased from Heungnong Seedling Co., Ltd., and tomato blight was Phytophthora infestans (Mont.) De Bary. 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. Pathogen Phytophthora infestans PIT strains were inoculated in oatmeal agar and incubated for 7 days in a dark state at 20 ° C. incubator for 7 days while irradiating with light for 16 hours a day to form sporangia. I was.

By spraying a microbial culture of Ahn004 strain on Seedling tomato processing of a medicament and air drying of that after, the addition of sterilized distilled water to the culture medium and harvesting the yujujanang and 3x10 ⁴ per ml to examine the blood cells to step spore concentration under a light microscope one yujujanang for 1 day The suspension was made at low temperature after the suspension was discharged, and then sprayed onto the treated tomato seedlings. Tomato seedlings inoculated with the pathogen were incubated for 1 day in a 20 ° C. chamber, and then placed in a constant temperature and humidity chamber (70% relative humidity) for 2 days. After examining the area ratio of the lesions formed in each tomato seedling, 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 medium (containing microbial intracellular material) of the bacteria Ahn004 (KACC91401) belonging to the genus Arthrobacter sp. Cultured by the method described in Example 1 were compared with the untreated group (FIG. 3). It was. In general, when a tomato late blight is caused by Phytophthora infestans , the necrosis of the tomato leaves is wilted. Referring to Figure 3, in the treatment-free (left) tomato infestation caused by the phytophthora infestans inoculated, the tomato leaves withered, and the culture treatment of Ahn004 bacteria (right) due to the bactericidal effect on the Phytophthora infestans Tomato leaves are healthy with no wilting symptoms.

As a result, when the culture solution of the fungus Ahn004 (KACC91401) belonging to the genus Athrobacter sp. ( Arthrobacter sp.) Was treated, tomato pest control effect was 95%. As a control drug, 100ppm of chlorothalonil was used, which showed 100% control value.

The microbial agent of the present invention can be prepared in the form of various preparations, including liquids, hydrates, powders, granules, emulsions, and the like, including the cultures or cultures of the strains or strains of the invention. The manufacturing method is according to the general manufacturing method in the art.

1 is a result of culturing in a novel Arobacter genus Ahn004 strain in Bennet medium and observed with a scanning electron microscope (Scanning Electron Microscope).

Figure 2 shows the results of 16S rRNA gene analysis of novel Arobacter genus Ahn004.

Figure 3 is a photograph showing the results of tomato late blight control effect by the treatment of the culture medium of the Acrobacter genus Ahn004, tomato treated with the culture medium of the Atrobacter Ahn004 and infected with the tomato late blight (right) and not treated (Left) is the result of infecting tomato blight without.

<110> Konkuk University Industrial Cooperation Corp. <120> A novel Arthrobacter sp. and biopesticide comprising the strain          against tomato late blight <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 1378 <212> DNA <213> Artificial Sequence <220> <223> 16s rDNA <400> 1 agagtttgat cctggctcag gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60 gatgatccca gcttgctggg ggattagtgg cgaacgggtg agtaacacgt gagtaacctg 120 cccttaactc tgggataagc ctgggaaact gggtctaata ccggatatga ctcctcatcg 180 catggtgggg ggtggaaagc tttattgtgg ttttggatgg actcgcggcc tatcagcttg 240 ttggtgaggt aatggctcac caaggcgacg acgggtagcc ggcctgagag ggtgaccggc 300 cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca 360 caatgggcgc aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa 420 acctctttca gtagggaaga agcgaaagtg acggtacctg cagaagaagc gccggctaac 480 tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat attgggcgta 540 aagagctcgt aggcggtttg tcgcgtctgc cgtgaaagtc cggggctcaa ctccggatct 600 gcggtgggta cgggcagact agagtgatgt aggggagact ggaattcctg gtgtagcggt 660 gaaatgcgca gatatcagga ggaacaccga tggcgaaggc aggtctctgg gcattaactg 720 acgctgagga gcgaaagcat ggggagcgaa caggattaga taccctggta gtccatgccg 780 taaacgttgg gcactaggtg tgggggacat tccacgtttt ccgcgccgta gctaacgcat 840 taagtgcccc gcctggggag tacggccgca aggctaaaac tcaaaggaat tgacgggggc 900 ccgcacaagc ggcggagcat gcggattaat tcgatgcaac gcgaagaacc ttaccaaggc 960 ttgacatgga ctggaaatac ctggaaacag gtgccccgct tgcggtcggt tcacaggtgg 1020 tgcatggttg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa 1080 ccctcgttct atgttgccag cgcgttatgg cggggactca taggagactg ccggggtcaa 1140 ctcggaggaa ggtggggacg acgtcaaatc atcatgcccc ttatgtcttg ggcttcacgc 1200 atgctacaat ggccggtaca aagggttgcg atactgtgag gtggagctaa tcccaaaaag 1260 ccggtctcag ttcggattgg ggtctgcaac tcgaccccat gaagtcggag tcgctagtaa 1320 tcgcagatca gcaacgctgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtc 1378 <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)

Atobacter oxydans strain having a control effect against Phytophthora infestans . The strain of claim 1, wherein the strain is Atrobacterium Ahn004 (KACC91401). The strain according to claim 1 or 2, wherein the strain has a control activity against tomato or potato late blight. A composition for preventing or treating tomato or potato late blight infections comprising the strain of claim 1 or 2 and an agrochemically acceptable carrier. A method of preventing or treating an infection of tomato or potato late blight by treating the strain of claim 1 or 2 or a culture thereof to a plant site or soil. A method for preventing or treating infection of tomato or potato late blight by treating the composition of claim 4 to plant parts, or soil.

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