KR101279044B1 - Pantoea dispersa WCU35 strain, composition for control plant disease and control method of plant disease with same - Google Patents

Abstract

The present invention relates to a novel Pantoea dispersa WCU35 strain, a composition for controlling plant diseases containing the same, and a method for controlling plant diseases, the novel Pantoea dispersa according to the present invention. By spraying or irrigation of WCU35 strains on plants, they are not only able to control various plant diseases biologically, but also induce resistance (ISR) that activates immune function on the plants themselves, which can be applied to multiple crops simultaneously. In crops, it is possible to simultaneously exert effects on various diseases, and the economical substitution effect of using microorganisms is large and has a high performance, thereby increasing the marketability and productivity of crops.

Description

Pantoia dispersa WC35 strain, composition for controlling plant diseases and method for controlling plant diseases {Pantoea dispersa WCU35 strain, composition for control plant disease and control method of plant disease with same}

The present invention relates to Pantoia dispersa WCU35 strain, a composition for controlling plant diseases containing the same, and a method for controlling plant diseases.

Pesticides and fertilizers, the products of modern science, have greatly contributed to the liberation of humankind from hunger by increasing agricultural productivity. Recently, however, there has been a great deal of social attention on negative aspects such as phosphorus and livestock toxicity and environmental pollution caused by misuse and residue of pesticides and fertilizers, rather than the positive aspects of synthetic pesticides and fertilizers. With the advent of the well-being era, there is an increasing demand for eco-friendly agricultural products in which synthetic pesticides are used or crops are grown without pesticides. In addition, farmers are also importing a lot of low-priced foreign agricultural products by import liberalization, and as an alternative, organic agricultural products with a high income per unit area are preferred.

However, it is very difficult to harvest crops without controlling plant diseases when crops are grown with environmentally friendly farming methods in actual packaging. Studies show that peaches and apples lost 100% and 97%, respectively, and vegetables such as cucumbers, cabbages, and tomatoes lost between 39 and 63% when grown without crop protection. Therefore, in order to produce eco-friendly agricultural products, there is a need for biopesticides having excellent effects that can replace synthetic pesticides.

Biopesticides include microbial pesticides that control plant diseases, pests and weeds using microorganisms, and biochemical pesticides that use natural products produced by plants or microorganisms. In the case of microbial pesticides, it is difficult to mass-produce and formulate products, and the preservation stability of the products is low, and thus the market is not greatly expanded because standardization, standardization, and ease of distribution are not secured like chemical pesticides. In addition, when the microbial pesticide is used for the actual packaging, the plant disease can be controlled only after the microorganisms have grown in a significant amount after treatment, and thus there is a difficulty in controlling the plant disease in a short time. In addition, because the spectrum of the applied pests are narrow, various biopesticides have to be treated in order to control other plant diseases occurring in crops, which is a real economic burden for farmers.

In addition, in the past, microorganisms, which mainly have antimicrobial activity against pathogens, have been used for biological control, but recently, resistance reactions induced in crops by inoculating microorganisms in advance with the purpose of inducing resistance to diseases in crops ( Induced Systemic Resistance ("ISR") is used. The latter method has been evaluated as a very desirable control method in that the control effect is long lasting in addition to the advantage that it does not cause problems such as environmental pollution, weakness and residual toxicity.

Meanwhile, Pantoea Dispersa dispersa ) Bacteria are separated and cultured from bumblebees, and it is confirmed that they have pathogenicity to bumblebees. Therefore, many studies are underway in the development of pollen-mediated insects (Kwon, Won-Tae et al. -20), Pantoea dispersa (wheat cultivation) dispersa ) has been reported to improve the growth and resistance of wheat to the treatment of bacteria (G. Selvakumar et al., World Journal of Microbiology and Biotechnology Volume 24, Number 7 955-960 ISSN 1573-0972).

However, the control effect against plant pathogens has not been confirmed so far.

 Won-Tae Kim et al., Journal of The Korean Beekeeping Society, Volume 23, Issue 1, Issue 45 (April 2008) pp.13-20 ISSN 1225-0252 Listed KCI  G. Selvakumar et al., World Journal of Microbiology and Biotechnology Volume 24, Number 7 955-960 ISSN 1573-0972

The present inventors, Pantoea dispersa ( Pantoea) isolated from the soil and leaves of plants dispersa ) By spraying or irrigation of WCU35 strains on plants, they can not only control various plant diseases biologically, but also induce resistance to activate immune function on plants themselves, and have excellent control effects against various plant diseases. It was confirmed that the present invention was completed.

An object of the present invention is to provide a novel Pantoea dispersa WCU35 strain and its culture production method having excellent control against various plant pathogens.

Still another object of the present invention is to provide a plant disease control composition containing the Pantoea dispersa WCU35 strain and its culture.

Still another object of the present invention is to provide a method for controlling plant diseases using the composition for controlling plant diseases.

In order to achieve the above object, the present invention provides a Pantoea dispersa WCU35 strain (KACC91642P) having a control activity against plant pathogens.

Pantoea dispersa WCU35 strain (KACC91642P) according to the present invention is Magnaporthe oryzae , Thanatephorus cucumeris , Botrytis cinerea , Pie Saratov Shoot inpe Afghanistan's (Phytophthora infestans), Puccini Oh rekon dita (Puccinia recondita), blue Almeria Grammy Nice form Spanish system Hor Day (Blumeria graminis f. sp. hordei ), Colle sat tree Colchicum Coco death (Colletotrichum coccodes), pastries It is characterized by having control activity against at least one plant pathogen selected from Phytophthora capsici and Xanthomonas campestris pv.vesicatoria .

Pantoea dispersa according to the present invention ( Pantoea) dispersa ) WCU35 strain (KACC91642P) or a composition containing the culture thereof is rice blast (pathogen: Magnaporthe oryzae ), rice leaf blight (pathogen: Thanatephorus cucumeris ), wheat red rust disease (pathogen: Puccinia recondita ), pepper anthrax (pathogen: Colletotrichum coccodes ), barley powdery mildew (pathogen: Blumeria graminis f. sp. hordei ), tomato gray mold (pathogen: Botrytis cinerea ), tomato late blight (pathogen: Phytophthora infestans ), pepper late blight (pathogen: Phytophthora capsici ), pepper bacterial spot disease (pathogen: Xanthomonas campestris pv. vesicatoria ) is characterized by having a control activity against at least one plant disease selected from.

Hereinafter, the present invention will be described in detail.

The present invention, pantoea dispersa ( Pantoea) having a control activity against plant pathogens dispersa ) provides the WCU35 strain (KACC91642P).

More specifically, Pantoea dispers ( Pantoea) according to the present invention dispersa ) The WCU35 strain was isolated from the soil and leaves of the plant. As a result of analyzing the sequencing of the 16S rDNA of the strain, it was confirmed that Pantoea dispersa . The novel strain is Pantoea dispersa ( Pantoea dispersa ) was designated as WCU35 strain, and was deposited with the National Agricultural Science Institute Agricultural Genetic Resource Center on April 22, 2011 and was given accession number KACC91642P. See FIGS. 1 and 2.

The present invention is Pantoea dispersa ( Pantoea dispersa ) provides a culture of the WCU35 strain (KACC91642P) and a method of producing the same.

Pantoea dispersa of the present invention ( Pantoea) dispersa ) WCU35 strain can be cultured in a common medium for bacterial growth, such as Tryptic soybean broth liquid medium or Tryptic soybean agar.

In addition, while general bacteria grow well at relatively high temperatures of about 30 ° C. to 40 ° C. and do not grow well when the temperature is low, Pantoea dispersa according to the present invention ( Pantoea) dispersa ) The growth temperature of the WCU35 strain is characterized by being well cultured even at 20 to 30 ° C., and has a great advantage as a biological control agent for plant disease control considering the onset temperature of major plant diseases is 20 to 25 ° C.

The present invention is Pantoea dispersa ( Pantoea dispersa ) WCU35 strain (KACC91642P) or the Pantoea dispersa ( Pantoea) dispersa ) Provides a composition for controlling plant diseases, containing the culture of WCU35 strain (KACC91642P).

In addition, Pantoia dispers ( Pantoea) according to the present invention dispersa ) Since the WCU35 strain shows no pathogenicity against crops such as rice, tomato, wheat, barley and red pepper, it can be used as a biological control agent.

More specifically, Pantoia dispers (Pantoea dispersa) WCU35 strain is 1 × 10⁵ cfu / ml to 1 × 10¹² Rice blast disease by spraying the culture dilution solution containing the concentration of cfu / ㎖ to the plantMagnaporthe grisea), Rice leaf blight (pathogen:Thanatephorus cucumeris), Wheat red rustPuccinia recondita), Pepper anthrax (pathogens:Colletotrichum coccodes), Wheat flour (pathogen:Blurmeria graminis f. sp.hordei), Tomato gray mold (pathogen:Botrytis cinerea), Tomato late blight (pathogens:Phytophthora infestans), Pepper blight (pathogens:Phytophthora capsici), Pepper bacterial spot disease (pathogen:Xanthomonas campestris pv.vesicatoriaIt is effective in controlling plant diseases such as rice blast, tomato gray mold, wheat red rust, pepper anthrax, and pepper bacterial spot pattern disease, especially rice blast and tomato ash mold. I could confirm it.

Pantoea dispersa according to the present invention ( Pantoea) dispersa ) Spray or irrigate the WCU35 strain itself, its culture, or a composition comprising an extract of the culture in an amount capable of inhibiting plant diseases on the environment in which the plant is growing (e.g. soil, water) or on the surface of a growing plant. As a result, various plant diseases can be biologically controlled. At this time, if necessary, it can be used as a composition for controlling plant diseases by mixing with a carrier commonly used in the field of pesticides and formulated in powder, pellets, granules or solutions. The carrier may be water, white carbon, kaolin, and the like, and the culture may be a liquid culture or a solid culture.

The plant disease is any one or more selected from rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley flour, pepper anthrax and pepper bacterial spot disease, more preferably rice blast, tomato grayish At least one selected from fungal disease, red pepper anthrax, wheat red rust and red pepper bacterial spot pattern disease, is applied to crops in a conventional manner.

The novel Pantoea dispersa WCU35 strain (KACC91642P) according to the present invention can not only biologically control various plant diseases by spraying or irrigation on plants, but also can activate immune functions on the plants themselves. Induced resistance (ISR) is induced, can be applied to multiple crops at the same time, there is an advantage that can be effective at the same time for multiple diseases in one crop.

The novel Pantoea dispersa WCU35 strain (KACC91642P) according to the present invention has a high economical replacement effect by using microorganisms and has a high performance, thereby increasing the commercialization and productivity of crops. .

Figure 1 shows the nucleotide sequence of the novel Pantoea dispersa WCU35 strain (KACC91642P) 16S rDNA according to the present invention,
Figure 2 is a result confirming the phylogenetic position through the 16S rDNA of the novel Pantoea dispersa WCU35 strain (KACC91642P) according to the present invention,
3 is a result confirming the control effect against plant pathogens using the novel Pantoea dispersa (WCC35 strain) (KACC91642P) according to the present invention,
(RCB: Rice Blast, RSB: Rice Leaf Blight, TGM: Tomato Blight, TLB: Tomato Blight, RPA: Chilli Anthrax, WLR: Wheat Red Rust)
Figure 4 is a result confirming the induced resistance (induced resistance) to the novel Pantoea dispersa WCU35 strain (KACC91642P) according to the present invention.
(0: no infection, 1: less than 5% infection, 2: less than 10% infection, 3: less than 20% infection, 4: less than 30% infection, 5: more than 30% infection)

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Example 1 Isolation and Identification of Pantoia dispersa WCU35 Strain

(1) Molecular systematic analysis of strain

Strains with excellent antimicrobial activity were selected from the microorganisms isolated from the roots of Dongjin rice on the farm in Chonnam National University.

For molecular analysis of the selected strains, first, 16S rDNA sequencing was performed. As a result, the nucleotide sequence of FIG. 1 (SEQ ID NO: 1) was confirmed, and the homology search was performed with the sequence database of NCBI GeneBank, and the systematic location was identified using the CLUSTAL X program and the PHYLIP program.

Molecular systematic analysis based on the 16S rDNA sequence of the selected strains, as can be seen in Figure 2, as a strain belonging to the phylogenetic group including the species of the genus Pantoia as Pantoea dispersa and 99 It was confirmed that the flexibility relationship was more than%.

The isolated and selected strains were named Pantoea dispersa WCU35, and were deposited on April 22, 2011 at the National Institute of Agricultural Science, Agricultural Research and Development Institute, and were assigned accession number KACC91642P.

Example 2 Control Effect of Pantoia dispersa WCU35 on Plant Disease

The plant diseases (plant pathogens) of Table 1 were assayed for assaying control effects against various plant diseases using the culture medium of the selected Pantoea dispersa WCU35 strain.

In order to prepare the crops for the experiment, put a soil top for rice and a garden top for tomatoes, cucumbers, peppers, wheat and barley in a disposable plastic pot with a diameter of 4.5 cm. Varieties: Seeds of Seogwang Tomato, Heung-Nong Seedling), Red Pepper (Bujiang, Heung-Nong Seedling), Wheat (Breed: Joung-Gwang), and Barley (Great: East Barley) are sown in each pot, and then in greenhouse (25 ± 5 ℃). Three to four leafy rice, two to three leafy tomatoes, three to four leafy peppers, one leafy wheat and barley were prepared for the experiment.

The screened Pantoea dispersa WCU35 was prepared using TSB (Tryptic soybean broth) liquid medium at 28 ° C., 200 rpm, 48 hours, or TSA (Tryptic soybean agar). After culturing, the strain obtained after the cultivation was used at a concentration of 0.8 to 1.5 at OD 600 nm of absorbance with sterile water using a UV-Visible Spectrophotometer (Shimadza, Japan).

A 3-fold dilution of the culture solution of the Pantoea dispersa (WCU35) strain obtained above was treated by foliar spraying on rice, tomato, pepper, wheat, and barley prepared one day before inoculation of the plant pathogen of Table 1 above. After 7 days of treatment, the control effect on various plant diseases was calculated as the control value (%) according to Equation 1 below.

(1) Test for Rice Blast (RCB)

Magnaporthe grisea (Hebert) Barr strain (obtained from Korea Research Institute of Chemical Technology) was inoculated into rice bran agar (20 g of rice bran, 10 g of dextrose, 15 g of agar, and 1 L of distilled water). Incubated for 2 weeks in the incubator. Pathogen-grown media was rubbed with a Rubber Polishman to remove the media hyphae, and spores were formed for 48 hours on a fluorescent lighted shelf (25 ± 2 ° C.). The formed conidia were sprayed sufficiently to flow into the plant after making a concentration of spore suspension (5 × 10 ⁸ spores / ml) with sterilized distilled water. Inoculated rice was treated in a dark state for 24 hours (relative humidity 100%, 25 ± 5 ℃), and then placed for 5 days in the growth temperature of 25 ± 5 ℃, relative humidity of 70-80% and then examined the lesion area ratio.

(2) Test for Rice Sheath Blight (RSB)

Put an appropriate amount of bran into a 1 L culture bottle and sterilize it. Thanatephorus cucumeris ) strain (obtained from Korea Research Institute of Chemical Technology) was inoculated and incubated in a 25 ° C. incubator for 7 days. The cultured mycelium masses were finely ground and inoculated evenly in a pot where rice was grown, and after 1 day of incubation in a humidified state (25 ° C), they were placed in a constant temperature / humidity chamber having a relative humidity of 80% or higher for 4 days to investigate disease occurrence. The incidence survey was to investigate the proportion of diseased area to seedling leaf area to the area of leaf area.

(3) Test for tomato gray mold (TGM)

Botrytis cinerea strain (obtained from Korea Research Institute of Chemical Technology) was inoculated into potato agar medium and incubated for 5 days in a 20 ℃ thermostat (cancer state). Spores were formed by incubating for another 7 days while crossing. Spores formed on the medium were harvested with potato dextrose broth medium (composition: 4 g of potato starch per 1 L, dextrose 20 g; purchased from Becton & Dickinson) and the spore concentration was 5 × 10 using a hemocytometer. Adjusted to ⁵ spores / ml and sprayed tomato seedlings. Inoculated seedlings were induced for 3 days in a 20 ° C wetland (95% relative humidity), and the lesion area ratio was examined.

(4) Test for Tomato Late Blight (TLB)

Pathogen Phytophthora infestans strain (obtained from Korea Research Institute of Chemical Technology) was put into oatmeal agar medium (composition: 60 g of oatmeal per 1 L, 12.5 g of agar; purchased by Beckton & Dickinson) Inoculated and incubated at 20 ° C. incubator. The spermatozoa formed in the culture medium was harvested by adding sterile distilled water, and the spores concentration was examined by using a hemocytometer under an optical microscope to produce 1 × 10 ⁵ spores / ml spore suspension, which was then subjected to low temperature treatment, and then sprayed with tomato seedlings. . The tomato seedlings inoculated with the pathogen were incubated for 1 day in a 20 ° C. chamber, and placed in a constant temperature and humidity chamber (70% relative humidity) for 2 days.

(5) Test for Wheat Leaf Rust (WLR)

The pathogen, Puccinia recondita Rob ex Desm (obtained from Korea Research Institute of Chemical Technology), is a live parasite and used as a seeding inoculation of wheat seedlings while passing directly to plants in the laboratory. Spore suspension (spore 0.67 g / L) was prepared with harvested spores and then sprayed with wheat seedlings. Seedlings were incubated for 1 day in a 20 ° C wet room and then transferred to a 20 ° C constant temperature and humidity room with a relative humidity of 70% to induce the onset, and the lesion area ratio was examined 7 days after inoculation.

(6) wheat flour ( Barley Powdery Mildew , BPM For

The pathogen Blumeria Graminis form spesis holday graminis DC f. sp. hordei Marchal) (Acquired from Korea Research Institute of Chemical Technology) is a live parasite, and was used as an inoculator for powdery mildew spores formed by artificially inoculating barley seedlings in a laboratory. Inoculated barley seedlings were placed in a constant temperature and humidity room at 20-23 ° C. and a relative humidity of 70% for 7 days, and then the lesion area ratio was examined.

(7) pepper anthrax ( Red Pepper Anthracnose , RPA For

Colletotrichum , the pathogen spores formed by inoculating coccodes ) strain (obtained from Korea Research Institute of Chemical Technology) into oatmeal agar medium and harvested with sterile distilled water, and examined spore concentration with blood cell under an optical microscope to make 3 × 10 ⁵ spores / ml spore suspension. Seedlings were sprayed. Pepper seedlings inoculated with pathogen were incubated for 2 days in a 25 ° C wet room, and placed in a constant temperature and humidity room (70% relative humidity) for 1 to 2 days.

(8) pepper plague ( Red pepper blight , RPB For

Phytophthora , a pathogen, capsici ) strain (obtained from Korea Research Institute of Chemical Technology) was inoculated into oatmeal agar medium and harvested spores with sterile distilled water, and spore concentration was measured by blood cell system under optical microscope to make 2 × 10 ⁵ spores / ml spore suspension. After irrigation, the cells were treated for 1 day at 28 ° C. in a 1 day to 2 days in 28 ° C. constant temperature and humidity room (relative humidity of 70%), and the lesion area ratio was investigated.

Pantoea for each plant disease dispersa ) The control effect of the WCU35 strain is shown in Table 2 below.

As can be seen in Table 2 and Figure 3, Pantoea dispersa WCU35 strain showed an excellent control effect of more than 60% against rice blast and tomato gray mold, more than 40% against wheat rust It was confirmed that the control effect, pepper anthrax disease showed more than 20% control effect. On the other hand, tomato blight and rice leaf blight were found to be less than 15% control effect.

Therefore, it was confirmed that Pantoea dispersa WCU35 strain can effectively control wheat rust disease, tomato gray mold, especially rice blast disease.

[ Example  3] Pantoia Dispersa WCU35 Inductive Resistance Test of

Xanthomonas causes bacterial spots on peppers to verify resistance to plants campestris pv. vesicatoria ) was inoculated artificially to pepper to test for induction of disease resistance.

Pepper seeds were sown in 9cm × 8cm pots with topsoil 10 days after sowing. When 3-4 leaflets were released, a suspension of Pantoea dispersa WCU35 strain was added to 1 × 10 ⁸ to 10 ⁹ cfu / ml. After diluting with, the roots of the pepper were irrigated about 5 ml. Five days later, the Xanthomonas campestris pv.vesicatoria pathogen, which was incubated for two days in a PDA (Petato dextrose agar), was sterile water containing 0.85% NaCl and 0.02% siloxane signature. After diluting with 1 × 10 ⁸ to 10 ⁹ cfu / ml, the pathogens were inoculated by spraying about 2 ml per plant. Plants inoculated with the pathogen were placed in a greenhouse (about 25 ° C.) for 10 days and then examined for leaf incidence.

As a result it can be seen in Figure 4, Pantoea dispersa according to the present invention ( Pantoea dispersa ) As a result of assaying resistance inducing resistance to bacterial spot pattern against WCU35 strain, it can be confirmed that the control effect is excellent.

National Academy of Agricultural Science KACC91642P 20110422

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Claims (10)

At least one selected from Magnaporthe oryzae , botrytis cinerea , Puccinia recondita , and Xanthomonas campestris pv.vesicatoria Pantoea dispersa WCU35 strain (KACC91642P), which has control activity against plant pathogens.
delete Cultured in tryptic soybean broth liquid medium or tryptic soybean agar according to claim 1, Magnaporthe oryzae , botrytis cinerea , Puccini Oh rekon dita (Puccinia recondita) and janto Pseudomonas cam paste less blood V Bezier Katori O board toy having a first antifungal activity against plant pathogens, or more species selected from (Xanthomonas campestris pv. vesicatoria) display peosa (Pantoea dispersa) WCU35 Culture production method of strain (KACC91642P). From Magnaporthe oryzae , botrytis cinerea , Puccinia recondita and Xanthomonas campestris pv. Vesicatoria according to claim 3 Culture of Pantoea dispersa WCU35 strain (KACC91642P) having control activity against at least one selected plant pathogen. Magnaporthe oryzae , Botrytis cinerea, containing cultures of Pantoea dispersa WCU35 strain (KACC91642P) or Pantoea dispersa WCU35 strain (KACC91642P) ( Botrytis cinerea ), Puccinia recondita ( Puccinia recondita ) and Xanthomonas campestris pv. vesicatoria A composition for controlling plant diseases having a control activity against at least one plant pathogen selected from.
delete 6. The method of claim 5,
The plant disease is a plant disease control composition, characterized in that at least one selected from rice blast, tomato gray mold, wheat red rust and red pepper bacterial spot disease.
delete A method for controlling plant diseases, comprising treating the control composition according to any one of claims 5 and 7 to a crop or an environment in which the crop is growing. The method of claim 9,
The control method is Pantoea dispersa ( Pantoea dispersa ) WCU35 strain (KACC91642P) is a method for controlling plant diseases, characterized in that the treatment of 1 × 10 ⁵ cfu / ㎖ to 1 × 10 ¹² cfu / ㎖.

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