KR20100085757A - A novel arthrobacter sp. ahn003 having protecting ability for rice blast disease - Google Patents

Abstract

PURPOSE: A noval Arthrobacter sp. Ahn003 and a biopesticide formulation containing the same are provided to enhance prevention effect from rice blast disease over a conventional chemical. CONSTITUTION: An Arthrobacter Ahn003 strain(KACC91400) has an effect of preventing rice blast disease. The strain has an activity against Magnaporthe grisea. A composition for preventing or treating rice blast disease contains Arthrobacter Ahn003 strain(KACC91400) and agriculturally acceptable carrier.

Description

A novel Arthrobacter sp. A novel strain of AHOB003 having control effect against rice blast disease. Ahn003 having protecting ability for rice blast disease}

The present invention relates to a novel Arobacter genus Ahn003 strain (KACC91400) strain having a control effect against rice blast, a method for controlling rice blast using the strain, and a biochemical pesticide preparation containing the strain.

According to the United Nations Food and Agriculture Organization (FAO) statistics, rice loses about 25% of its production by plant diseases, pests and weeds.

Types of rice-related diseases include blight leaf blight, leaf blight blight, stalk blight, tall legs, and heat blight, among which blight blight is a disease that causes much damage to rice production in Korea. Plague is caused by Magnaporthe grisea and occurs during the entire growing season of rice, including leaves, nodes, earwoods, ear branches, and rice seeds. The onset forms a dark greenish brown pattern in the case of leaves, which later become long ovals, and inhibits the growth of the whole. It is characterized by high humidity and high temperature at 25 ~ 28 ℃, the occurrence of many diseases, the formation of conidia which causes the spread of the disease, and once spread, it spreads rapidly around. Rice blasts are already causing a lot of losses in major rice producing regions in Southeast Asia, including Japan, so blast control is closely related to increased production. (J. of Plant Physiology 165 (2008) 114-124) The use of pesticides is the easiest way to control blasts that have a tremendous impact on rice production. Currently, as a fungicide for controlling febrile disease, tricyclazole and blasticidin-S are used. In recent years, however, the safety of pesticide use, the residual problems of crops and soils, and environmental and biological problems on surrounding ecosystems have been increasingly highlighted.

In order to improve these problems, the use of pesticides should be reduced, but the reality of reduced rice production cannot be overlooked, so many researchers have tried to control the use of safe natural products and biological control methods using microorganisms with little impact on crops and the external environment. Is being studied.

Biopesticides include microbial pesticides using bacteria, fungi and viruses, chemicals derived from microorganisms or plants, and plant incorporated protectants (PIPs) using gene mixtures. Among them, biopesticides are increasing in use due to their price competitiveness and low resistance. (Microbial biopesticides: an introduction.I: Micorbial biopesticides. London: Taylor and Francis, 2002, 1-12) In addition, microbial pesticides use microorganisms themselves to control pests and proliferate against plant pathogens that cause various diseases. Studies using antagonistic microorganisms that induce action, parasitic action, antibiotic action and phagocytosis are typical. Since 1961, Bacillus thuringiensis has been studied and utilized as a major target for biopesticides. (Daniel L. Sudakin, 2003, Biopesiticides, Toxical review, 22 (2), 83-90)

Controlling microbial pesticides in Korea has been reported to be active against rice blast, gray bacterium cabbage, and Candida, among which antimicrobial antibiotics are active. (Shim Kwan Kim, Chang Hwan Kim, 1989, Physiological Activity of Acetoxycycloheximide and its Strains, Journal of the Korean Society for Industrial Microbiology, 17: 307-312).

The present invention solves the above problems, and the object of the present invention as devised by the necessity of the above is to provide a novel strain having a control effect against rice blast.

Another object of the present invention is to provide a method for controlling rice blast using the strain.

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

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

In a preferred embodiment of the present invention, the strain is preferably an Atobacter Ahn003 strain (KACC91400), but is not limited thereto.

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

The present invention also provides a composition for preventing or treating rice blast infection, comprising the strain of the present invention and an agrochemically acceptable carrier.

In another aspect, the present invention provides a method for preventing or treating infection of rice fever by treating the strain of the present invention or a culture thereof or the composition of the present invention with a plant part 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 method for controlling rice blast, characterized in that using the strain.

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 the control of rice blast.

In the present invention, the microorganism having the control effect of rice blast refers to a microorganism having the ability to prevent the occurrence of rice blast or to cure a plant disease by treating the plant with the microorganism such as spraying the microorganism on the plant.

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 preventing and treating rice fever, which contains an effective amount of the genus Atobacter strain and comprises 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, 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 spraying liquid of the microorganism which is an active ingredient improve when the dispersion of a microbial pesticide composition is prepared by containing surfactant. 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 particular limitation regarding the method of using the microbial pesticide composition of the present invention, but for example, 1000 times, 2000 times, 4000 times, etc., dilution with water is made so as to obtain a predetermined dilution ratio determined at the time of pesticide registration, and the spraying solution is prepared, and the spraying thereof. Using a power sprayer, etc.

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

The strain of Abrobacterium Ahn003 of the present invention was deposited on October 07, 2008 under the name of the strain Ahn003 and accession number KACC91400 at the Agricultural Biotechnology Park of Korea (225-44, Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do). .

The invention is briefly described below.

The novel Acrobacter genus Ahn003 (KACC91400) of the present invention is an antimicrobial bacterium, characterized in that the gene nucleotide sequence of the 16S rRNA has a specific structure as follows. It has 98% homology with Arthrobacter oxydans and has excellent control effect in rice blast.

The present invention has the effect of providing a novel Arobacter genus Ahn003 strain (KACC91400) having a rice blast control effect, a rice blast control method using the same and a biochemical pesticide preparation containing the strain.

The strain according to the present invention is useful as a biopesticide because it exhibits a higher rice blast control effect than conventional chemical pesticides without destroying the ecosystem due to overuse that may occur in conventional chemical pesticides.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1.Isolation and Identification of Novel Arobacterium Ahn003 (KACC91400)

Culture medium

Bennet medium used for actinomycetes culture was used for suspension culture of the selected strain KACC91400. 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.

Morphological Identification

The purely isolated novel Arobacterium Ahn003 strain (KACC91400) was cultured in Bennet medium and observed in morphology with a scanning electron microscope (FIG. 1).

16S rRNA gene base sequence analysis

Genetic analysis of the novel strain Ahnobacter Ahn003 (KACC91400) was performed, and sequencing of the 16S rRNA gene was performed as follows. The suspension was cultured in Bennet medium for 7 days for DNA extraction, and then the cells were separated by centrifugation. DNA extraction was performed using Genomic DNA Extraction Kit (Intron).

PCR was carried out in the 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), 0.2 μg genomic DNA, 0.25 μM 16S rRNA gene primers as shown in Table 1 Amplification using primers. After PCR reaction (initial 94 ℃ 5 minutes once; 94 ℃ 1 minutes, 55 ℃ 1 minutes, 72 ℃ 1 minutes, 35 cycles; 72 ℃ 5 minutes once) and then amplified 16S rRNA gene portion of pGEM-T Easy vector ( After ligation to Promega co.), E. coli DH5α was transformed.

Transformed Escherichia coli was provoked in selection medium (1.5% agar, 50mg / L ampicillin, 100uM IPTG, 50mg / L X-gal) and incubated at 37 ° C for 12 hours. Insertion of the amplified 16S rRNA fragment into the vector was first selected as a blue / white colony, and only white colony was inoculated into LB medium and cultured. The culture was centrifuged for plasmid separation and Alkaline lysis was used. The isolated plasmid was digested with restriction enzyme Eco RI and confirmed insertion of amplified 16S rRNA fragment through electrophoresis.

The amplified 16S rRNA gene was analyzed by a sequencing device (ABI3100) and found to have the nucleotide sequence of SEQ ID NO: 1. The homology was confirmed using BLAST of the National Center for Biotechnology Information (NCBI) and as a result, Arthrobacter oxydans ) and 98% homology (Fig. 2).

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

Example 2 Rice Fever Control Effect of Abrobacterium Ahn003 Strain (KACC91400)

The rice varieties tested were Oryza sativa cv Nakdong, and the rice blast bacteria were Magnaporthe grisea and the KJ201 race was susceptible to rice cultivar Nakdong. Rice seeds were seeded and sown on aquatic soils (sub-farms), and the control effect was investigated using about 3-4 leaf rice plants prepared by cultivation for about 4 weeks in a greenhouse of 25 ± 5 ℃.

Rice blast bacteria were inoculated in potato juice medium (potato dextrose broth, PDB) and shaken at 25 ° C. for 7 days. Mycelium grown in liquid medium was cut using a Waring blender and sterilized in cooled rice bran medium (rice bran 20g, dextrose 10g, agar 2g, distilled water 1L), shaken well, poured into Petri-dish and hardened. Incubated. Spores formed by removing the aerial hyphae of the cultured bacteria and placed under a fluorescent lamp were harvested, and the concentration of spores was adjusted to 5 x 10 ⁵ per ml and used as an inoculum.

To investigate the control effect of the microbial culture, Tween 20 was added to the microbial culture to have a concentration of 250 mg / L, and the leaves were air dried for 24 hours. The rice leaves treated with the microbial culture solution were inoculated by spraying the spore suspension of rice blast fungus prepared above. Inoculated rice was incubated for 1 day after incubation at 25 ° C. incubation for 4 days in a constant temperature and humidity room at 25 ° C. and a relative humidity of 80%.

The results of treatment of the culture medium (containing microbial intracellular material) of the Atobacter genus Ahn003 strain (KACC91400) cultured by the method of Example 1 were compared with the untreated group (FIG. 3). In general, a severe rice infestation caused by Magnaporthe grisea causes the leaves to die. Referring to FIG. 3, both the untreated (right) and treated (left) cultures of the genus Ahn003 strain (KACC91400) were inoculated with Magnaporthe grisea . Was observed.

The control value which suppresses disease occurrence was calculated by the following formula.

As a result, when the culture solution of the genus Ahn003 strain (KACC91400) was treated, the rice blast control value was 80%. From this, it was confirmed that KACC91400 bacteria of the present invention has excellent rice blast control effect.

The biochemical preparations of the present invention can be prepared and used in the form of various preparations including the culture or culture of the strain of the present invention, that is, liquids, hydrates, powders, granules, emulsions and the like.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a scanning electron micrograph (Scanning Electron Microscope) of the novel Arobacter genus Ahn003 strain (KACC91400) strain.

Figure 2 shows the results of 16S rRNA gene analysis of novel Arobacter genus Ahn003 (KACC91400).

Figure 3 shows the rice (right) and the untreated rice (left) in which the blast was severely treated by treating the culture medium of the genus Ahn003 (KACC91400).

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Claims (6)

Atrobacter oxydans strain having a control effect against rice blast. The method of claim 1, wherein the strain is Genus Ahn003 strain (KACC91400). The strain according to claim 1 or 2, wherein the strain has a control activity against Magnaporthe grisea . A composition for preventing or treating rice blast infection, comprising the strain of claim 1 or 2 and an agrochemically acceptable carrier. A method of preventing or treating an infection of rice fever by treating the strain of claim 1 or 2 or a culture thereof with a plant part or soil. A method of preventing or treating an infection of rice fever by treating the composition of claim 4 with a plant part or soil.

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