KR101250020B1 - Simplicillium lamellicola KRICT3 strain, composition for control plant diseases and control method of plant diseases with same - Google Patents

Abstract

Simplicilium lamelicola KRICT3 strain according to the present invention is a microorganism native to Korea, economical substitution effect is large, has a high performance, and has excellent antibacterial activity against various plant diseases, the productability and productivity of crops There is an effect that can be increased.

Description

Simplicillium lamellicola KRICT3 strain, composition for control plant diseases and control method of plant diseases with same}

The present invention relates to a simple siliumum lamelicola KRICT3 strain, a composition for controlling plant diseases containing the same, and a method for controlling plant diseases using the same. More specifically, the present invention relates to an antimicrobial activity against various plant pathogens as an overlapping parasite against gray fungus. The present invention relates to a novel simlysilium lamelicola KRICT3 strain, a composition for controlling plant diseases containing the KRICT3 strain or a culture thereof, and a method for controlling plant diseases using the same.

Pesticides and fertilizers, the products of modern science, have greatly contributed to the liberation of humankind from hunger by increasing agricultural productivity. In recent years, however, social interest in phosphorus and livestock toxicity and environmental pollution due to misuse and residue of pesticides and fertilizers has been increasing, and the well-being age that emphasizes the quality of life has come to reduce the use of synthetic pesticides. B There is an increasing demand for eco-friendly agricultural products in which crops are grown without pesticides. In addition, since farmers are importing a lot of low-priced overseas agricultural products through liberalization of imports, the area of eco-friendly agricultural products is gradually increasing as they prefer organic farming with high income per unit area. However, to grow crops with environmentally friendly farming methods in actual packaging, it is very difficult to harvest produce without controlling plant diseases. Studies show that peaches and apples lost 100% and 97% of their crops without crop protection, while crops of cucumbers, cabbage, and tomatoes lost between 39 and 63%. Therefore, in order to produce eco-friendly agricultural products, biopesticides with excellent plant disease control effects are required to replace synthetic pesticides.

Biopesticides include microbial pesticides using microorganisms to control plant diseases, pests and weeds, and biochemical pesticides using natural products produced by plants or microorganisms. In the case of microbial pesticides, the microorganisms can be classified into antimicrobial producing microorganisms, duplicate parasitic microorganisms parasitic plant phytopathogens, and microorganisms which enhance induction resistance to plants, depending on the method of sterilizing the microorganisms. Among them, the phytopathogens, particularly fungi parasitic parasites are known Trichoderma genus, Gliocladium genus, Acremonium genus. With Trichoderma spp developing biological pesticides Trichoderma harzianum T-22, TH35, TH315, T-39, ATCC 20475 and IMI 206040 strains and Trichoderma viridae ATCC 20476 strain, Trichoderma polysporum IMI 206039, Trichoderma stromaticum , Trichoderma virens , Trichoderma viride, etc. And Cliocladium catenulatum ( Clonostachys rosea f. catenulate identical name) is registered and commercially available under the trade names Prestop. In addition , various Acremonium spp strains are known that overlap parasitic fungi of various phytopathogenic fungi, such as Heminthosporium . However, there have been few reports on how to control plant diseases using the redundant parasitic microorganism Simplicillium lamellicola .

Therefore, the present inventors have discovered a novel Simplicillium lamellicola strain for the first time as a microorganism native to Korea, which has not yet been reported, and has parasitic pathogenic fungi and excellent control against plant diseases. The present invention has been completed.

An object of the present invention is to provide a novel Simplicillium lamellicola strain and its culture having excellent antibacterial activity against various plant diseases.

Another object of the present invention is a plant composition for controlling plant diseases containing the Simplicillium lamellicola strain or its culture, which has a high economical replacement effect and high performance as a microorganism native to Korea To provide a control method.

In order to achieve the above object, the present invention has excellent antimicrobial activity against a variety of plant diseases, novel Simplicillium lamelicola ( Simiplicillium lamellicola ) strains and cultures thereof.

The present invention is the simplicillium lamelicola ( Simiplicillium lamellicola ) provides a composition for controlling plant diseases and a method for controlling the same, containing the strain or the culture thereof.

Simplicillium lamelicola according to the present invention lamellicola strain is a microorganism native to Korea, Botrytis cinerea ) is characterized by having antimicrobial activity against various plant pathogens as a duplicate parasite.

Simplicillium lamelicola of the present invention ( Simiplicillium) lamellicola strain is Alternaria alternata ) , Cochliobolus heterostrophus ), Magnaporthe oryzae ), Botrytis Cineri cinerea ) , Botrytis allii ), Sclerotinia sclerotiorum ), Colletotrichum Orbitulae orbiculare ), Phytophthora capsici ), Phytophthora infestans infestans ), Fusarium oxysporum , Pythium ultimum ), Pythium graminicola and Rhizoctonia solani ) having antimicrobial activity against at least one plant pathogen selected from AG1.

In addition, Simplicillium lamelicola of the present invention ( Simiplicillium) lamellicola ) strains and their cultures are in one or more plant diseases selected from the group consisting of rice blast, rice leaf blight, tomato blight, pepper blight, cucumber ash fungus, tomato ash fungus, lily blight and barley powdery mildew. It is characterized by having a control effect for.

Hereinafter, the present invention will be described in detail.

The present invention is gray mold fungus ( Botrytis Simplicillium , which has antimicrobial activity against plant pathogens as a duplicate parasite against cinerea lamellicola ) KRICT3 (Accession No .: KCTC 11765BP) strain and culture of the strain are provided.

More specifically, Simplicillium lamelicola ( Simiplicillium ) according to the present invention lamellicola ) KRICT3 strains from the surface of strawberries infected with gray mold ( Botrytis) Strains showing bactericidal activity against gray fungal pathogens as well as the characteristics of double parasitic bacteria parasitic on Ccinerea ) were screened.

As a result of analysis of the nucleotide sequence of the 28S rRNA gene (SEQ ID NO: 1) and the ITS region (SEQ ID NO: 2) for the identification of the strain, it was identified as not simplicity, but simplicity lamelicola, One strain was named Simplicillium lamelicola KRICT3, and was assigned the accession number KCTC 11765BP to the Korea Institute of Bioscience and Biotechnology, September 15, 2010. See FIGS. 6 and 7.

In the present invention, the plant pathogen is one or more plant diseases selected from the group consisting of rice blast, rice leaf blight, tomato blight, pepper blight, cucumber ash fungus, tomato ash fungus, lily blight and barley powdery mildew Characterized in having an excellent control effect for.

More specifically, the simplicillium lamelicola of the present invention ( Simiplicillium) lamellicola) replacement for the cause of a variety of plant diseases KRICT3 strain culture results, causing jeommunuibyeong various plants and Leah Alter Alter appear (Alternaria alternata ) , a corn germline bacillus , Cochliobolus heterostrophus ), The rice blast fungus Magna Forte Duck Party (Magnaporthe oryzae ), Botrytis cineri, a fungal pathogen of various plants cinerea ) , an onion ash fungus bacillus Botrytis allii), causing gyunhaekbyeong of various plants Klee's Nias Loti Crescent to Tio Rum (Sclerotinia sclerotiorum ), Colletotrichum orbiculare (cucumber anthrax), Phytophthora (capsicum blight) capsici ), Phytophthora , a tomato and potato plague pathogen infestans), wilt fungus is Fusarium oxy's forum (Fusarium oxysporum ), Pythium causes mosslock disease on various plants ultimum ), Pythium causing grass mozzarella graminicola ) and Rhizoctonia , a rice plant leaf blight pathogen solani ) was confirmed to effectively inhibit the growth of plant pathogens such as AG1. Please refer to Fig.

In particular, the simplicity lamelicola KRICT3 strain according to the present invention is alternata alternata (Alternaria alternata), Cocciliobolus heterostropus (Cochliobolus heterostrophus), Magna Forte Orissa (Magnaporthe oryzae), Botrytis CinemariBotrytis cinerea),Botrytis AliBotrytis allii), Sclerotinia screrothiorum (Sclerotinia sclerotiorum), Colletotricum OrbiculaleColletotrichum orbiculare) Was confirmed to have excellent bactericidal activity.

The present invention is simplicillium lamelicola ( Simiplicillium lamellicola ) KRICT3 (Accession No .: KCTC 11765BP) strain or Simplicillium lamelicola ( Simplicillium lamellicola ) Provides a composition for controlling plant diseases, containing a culture of KRICT3 (Accession Number: KCTC 11765BP) strain.

The composition for controlling the present invention is at least one plant disease selected from the group consisting of rice blast, rice leaf blight, tomato blight, pepper blight, cucumber gray mold, tomato gray mold, lily blight and barley powdery mildew. It is characterized by having an excellent control effect.

More specifically, the control effect on various plant diseases in the greenhouse experiment using the composition for the control was tested.

As a result, rice blast disease (pathogen: Magnaporthe oryzae ), rice leaf herb blight (pathogen: Rhizoctonia) solani AG1), tomato late blight (pathogen: Phytophthora infestans ), pepper late blight (pathogen: Phytophthora capsici ), cucumber and tomato gray mold disease (pathogen: Botrytis cinerea ), lily leaf blight (pathogen: Botrytis) elliptica ) and barley powdery mildew (pathogen: Blumeria graminis f. sp. hordei ) were found to show effective control against various plant diseases, including rice, tomatoes, cucumbers, wheat, barley, cabbage and red pepper. Was found to show no pathogenicity at all.

In the present invention, the composition for the control is simplicity lamicola ( Simplicillium lamellicola ) KRICT3 (Accession No .: KCTC 11765BP) strain is characterized in that it contains at a concentration of 1 × 10 ⁴ to 1 × 10 ⁹ spores / ml, more preferably 1 × 10 ⁶ to 1 × 10 ⁸ spores / ml It should be contained in the concentration of. Please refer to Fig.

The present invention is simplicillium lamelicola ( Simiplicillium lamellicola ) KRICT3 (Accession No .: KCTC 11765BP) strain or Simplicillium lamelicola ( Simplicillium lamellicola ) Provides a method for controlling plant diseases, characterized in that the composition for controlling plant diseases containing a culture of KRICT3 (Accession Number: KCTC 11765BP) strain is treated to the fruit or plantation of the crop.

In the present invention, the plant disease is one or more selected from the group consisting of rice blast, rice leaf blight, tomato blight, pepper blight, cucumber ash fungus, tomato ash fungus, lily blight and barley powdery mildew, Apply to crops in the usual way.

The control composition according to the present invention by a specific method is to spray on the soil, crop leaves, stems, flowers or berries, can be used by diluting the composition in water according to the density of the pathogens in the package, the onset environment, etc. during the packaging process. And, in order to add the antimicrobial activity of the microorganism strain of the present invention may further comprise a foliar spray or irrigation spray. In addition, during the spraying season, it is possible to further increase the control effect by prophylactically treating each plant pathogen before the onset and propagation.

Simplicilium lamelicola KRICT3 strain according to the present invention is a microorganism native to Korea, economical substitution effect is large, has a high performance, and has excellent antibacterial activity against various plant diseases, the productability and productivity of crops There is an effect that can be increased.

1 is a novel simplicity lamicola ( Simiplicillium) according to the present invention lamellicola ) shows the nucleotide sequence of the 28S rRNA gene of KRICT3 (Accession Number: KCTC 11765BP) strain,
2 is a novel simplicity lamicola ( Simiplicillium) according to the present invention lamellicola ) shows the nucleotide sequence of the ITS region of KRICT3 (Accession Number: KCTC 11765BP) strain,
Figure 3 is a photograph showing the flora of the novel Simplicillium lamellicola ( Simiplicillium lamellicola ) KRICT3 (Accession Number: KCTC 11765BP) strain according to the present invention,
4 is a novel simplicity lamicola ( Simiplicillium) according to the present invention lamellicola ) The result of replacement culture showing antimicrobial activity against various plant pathogens of KRICT3 (Accession No .: KCTC 11765BP) strain (the right side of Petri dishes is KRICT3 (Accession No .: KCTC 11765BP) strain),
(A: rice blast bacterium, B: corn stalk bacterium, C: ash fungus, D: spot fungus, E: wilting bacterium, F: cucumber anthrax, G: turf mozzarella, H: Pythium mozzarella, I : Rhizoctonia mozolox bacillus)
5 is a result confirming the control effect of lily leaf blight on the treatment of the concentration of the novel Simplicillium lamellicola ( Simiplicillium lamellicola ) KRICT3 (Accession Number: KCTC 11765BP) strain according to the present invention,
(A: untreated, B: 4.1 × 10 ⁵ spores / ml treated, C: 1.2 × 10 ⁶ spores / ml treated)
6 is a novel simplicity lamicola ( Simiplicillium) according to the present invention lamellicola ) is a result of confirming the homology of the nucleotide sequence for 28S rRNA gene of KRICT3 (Accession Number: KCTC 11765BP) strain,
7 is a novel simplicity lamicola ( Simiplicillium) according to the present invention lamellicola ) The homology of the nucleotide sequence to the ITS region of KRICT3 (Accession Number: KCTC 11765BP) strain was confirmed.

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 of Simplicillium lamelicola KRICT3 Strain

On the surface of the strawberry plastic house, which had severely developed gray mold disease, located in Nonsan, Chungcheongnam-do, countless cases of gray mycelia and spores were observed on the surface of strawberries infected with gray mold. A growing white mold was found.

Strawberry berries showing the above symptoms were collected and separated from them by white colonies. In order to select strains showing bactericidal activity against the gray fungus Botrytis cinerea , a replacement culture experiment was performed. Inoculate with a mycelial fragment (6.5 mm diameter) of the isolated strain on one side of potato agar medium of 8.5 cm diameter Petri dish and incubate for 3 days at 20 ° C. Incubated for 5 days at ℃. Strains forming the largest lowland in the replacement culture experiment were selected and named as KRICT3 strain.

Example 2 Identification of Simplicillium Lamelicola KRICT3 Strains

In order to identify KRICT3 strains selected in Example 1, ITS region and 28S rDNA sequencing were performed.

DNA was isolated from the mycelia of the isolated strain to obtain the nucleotide sequence of the 28S rRNA gene and the ITS region of the KRICT3 strain, followed by mixing with PCR-premix (PCR-premix; iNtRON Biotechnology, Korea) and LR0R (5'-ACC CGC). TGA ACT TAA GC-3 ') and LR 7 (5'-TAC TAC CAC CAA GAT CT-3') primers were used to amplify 28S rRNA genes and ITS 5 (5'-GGA AGT AAA AGT CGT AAC AAG G) -3 ') and ITS 4 (5'-TCC TCC GCT TAT TGA TAT GC-3') primers were used to amplify the ITS region.

The amplified PCR product was purified using a Wizard PCR prep kit (Wizard PCR prep kit; Promega, Medison, WI, USA), and then a base sequence was obtained by requesting a macrogen (Seoul, Korea). The base sequence was compared with GenBank database and base sequence using BLAST of NCBI. Sequences were sorted by CLUSTAL X and a neighbor-joining tree was created using PHYDIT program version 3.0.

The nucleotide sequence (SEQ ID NO: 1) and the nucleotide sequence (SEQ ID NO: 2) of the ITS region of the selected strain of the 28S rRNA gene were analyzed based on the neighbor-joining algorithm. As shown, the KRICT3 strain had a homology of 99.6% in the simprisilium lamelicola CBS 116.25 and the nucleotide sequence of the 28S rRNA gene.

In addition, the base sequence of the ITS region, as can be seen in Table 2 and FIG. 7, it was confirmed that the nucleotide sequence of the simprisilium lamelicola CBS 116.25 and the ITS region is a novel strain showing 99.2% homology.

From the above results, the strains selected in the present invention were not identical but were identified as Simlyillium lamellicola, and the novel strains selected were named Simplicillium lamellicola KRICT3, and the Korea Biotechnology Research Institute Bioresource Center 2010. On September 15, 2008, he was assigned accession number KCTC 11765BP.

[ Example  3] Simplicillium Ramelicola KRICT3  Strain Redundancy  Confirm

The flora of the novel KRICT3 strain isolated after isolation from the results of Examples 1 and 2 was white, and the flora of the gray mold fungus was gray.

In addition, the diameter of the mycelial fungal mycelium was examined using parasitic characteristics of the mycelial fungus of the KRICT3 strain using the characteristic of having a mycelial diameter more than 10 times higher than that of the KRICT3 strain. KRICT3 strains were inoculated into potato liquid medium (potato dextrose broth, Becton Dickinson & Co.) and shaken at 150 rpm for 10 days at 20 ° C. The cells were removed by filtration of KRICT3 culture medium with two-ply gauze, and the spore suspension harvested by centrifugation was diluted with potato liquid medium and 9 × 10 ⁴ , 3 × 10 ⁵ , 8 × 10 ⁵ , 3 × 10 ⁶ , Spore suspensions of 7 × 10 ⁶ , 2 × 10 ⁷ , 7 × 10 ⁷ , and 2 × 10 ⁸ concentrations were prepared.

Spores of gray fungal bacteria formed by culturing on potato agar medium were harvested with potato liquid medium and 1 × 10 ³ , 4 × 10 ³ , 1 × 10 ⁴ , 4 × 10 ⁴ , 1 × 10 ⁵ , 3 × 10 per ml, respectively. ^Five concentrations of spore suspensions were prepared. Into each well of a 96 well plate, 50 μl of each spore suspension of gray fungus and KRICT3 strain was shaken and cultured at 150 rpm for 7 days in a 20 ° C. incubator. The parasites of KRICT3 strains were investigated.

As a result, as can be seen in the results of Table 3, the simplicity lamella cola KRICT3 strain showed a white flora at most concentrations treated, it can be confirmed that the KRICT3 strain inhibits germination and growth of gray mold disease there was.

In the case of high concentration of spores of gray fungus and low concentration of spores of KRICT3 strain, the bacterial flora showed a grayish white flora, but it was confirmed that the two strains coexisted under the microscope. The hyphae that goes to mycelia of KRICT3, that is, mycelia were invaded and grown. Therefore, it could be confirmed from the above results that the novel S. similirium lamelicola KRICT3 strain of the present invention is a duplicate parasite against the gray mold.

Example 4 Bactericidal Effect of Simplicilium lamella coli KRICT3 Strain against Various Plant Pathogens

In order to confirm the bactericidal effect of S. similirium lamelicola KRICT3 strain against various phytopathogens, the mycelial fragment of KRICT3 strain on the one side of potato aextrose agar in Petri dish (8.5 cm diameter) 6.5 mm) was inoculated and then incubated at 20 ° C. for 3 days.

12 plant phytopathogens ( Rhizoctonia solani AG4, Pythium ultimum, Pythium graminicola, Phytophthora capsici, Colletotrichum orbiculare, Fusarium oxysporum, Sclerotinia sclerotiorum, Botrytis cinerea, Botrytis alliiae), Mag Alterporna Mycelial slices (diameter 8 mm) removed from the tip of the inocula were inoculated across the KRICT3 strain of potato agar medium precultured with the KRICT3 strain, Pythium ultimum , Pythium graminicola , Sclerotinia sclerotiorum , Botrytis cinerea , Botrytis allii at 20 ° C and the remaining phytopathogens Was incubated for 2 to 7 days at 25 ℃ to determine the size of the ring.

As can be seen in Table 4 and Figure 4, KRICT3 strains Rhizoctonia damping-off of the 12 species of plant pathogenic bacteria of the experiment Rhizoctonia solani (Rhizoctonia solani ) Alternaria with 11 different plant pathogens except AG4 alternata ) , Cochliobolus heterostrophus ), Magnaporthe oryzae , Botrytis Cineri cinerea ) , Botrytis allii ), Sclerotinia sclerotiorum ), Colletotrichum orbiculare , Phytophthora capsici ), Phytophthora infestans infestans ), Fusarium oxysporum ), Pythium ultimum , Pythium graminicola Graminicola ), such as phytopathogens were found to have bactericidal activity.

In particular, KRICT3 strains are Alternaria alternata , Cochliobolus Cochliobolus heterostrophus ), Magnaporthe oryzae ), Botrytis cineri ( Botrytis cinerea ) , Botrytis allii ), Sclerotinia sclerotiorum ), Colletotrichum orbiculare ) was confirmed to have excellent bactericidal activity.

[ Example  5] In greenhouse experiment Simplicillium Ramelicola KRICT3  Strain In plant diseases  Control effect

One) Simplicillium Ramelicola KRICT3  Six kinds of strain culture Plant disease  Control effect

Rice Blast Disease (Herogenus: Magnaporthe oryzae ) and Rice Leaf Blight ( Rhizoctonia ) by Treatment of Spore Suspension and Culture Filtrate of S. simillirium lamelicola KRICT3 Strain solani AG1), cucumber gray mold (pathogen: Botrytis cinerea ), tomato late blight (pathogen: Phytophthora infestans ), wheat rust (pathogen: Puccinia recondita ), and barley flour (pathogen: Blumeria) graminis f. sp. hordei ) and other plant diseases were tested in greenhouses.

In order to prepare the crops for the experiment, put the rice paddy soil for seeding of rice and the gardening soil for sowing of tomatoes, cucumbers, wheat and barley in a disposable plastic pot with a diameter of 4.5 cm. Seeds of cucumbers (variety: House bag tea, Eastern Hannong), tomato (variety: Seogwang Tomato, Monsanto Korea), wheat (variety: Eunpa wheat) and barley (variety: Eastern barley) in each pot, 25 ± 5 ° C.), 3-4 leaf rice, 2-3 leaf tomato, 1 leaf cucumber, wheat and barley prepared by cultivation for 1 to 4 weeks were used for the experiment.

Simplicirium lamelicola KRICT3 strains were inoculated in potato liquid medium (potato dextrose broth, Becton Dickinson & Co.) and shaken at 20 ° C. for 10 days at 150 rpm to obtain a culture solution. Filter the culture medium of the KRICT3 strain with a two-ply gauze to remove the mycelia, and centrifuged at 5000 rpm to harvest the supernatant (culture filtrate), add the culture solution and the same amount of sterile distilled water to the precipitate and shake well about 1 × 10 per ml ^Eight concentrations of spore suspensions were prepared. The spore suspension was sprayed on rice, tomato, cucumber, wheat and barley plants. The drug-treated plants were air-dried in a greenhouse for one day, and then inoculated with spores or mycelium fragments of each pathogen, and the plant diseases were investigated.

The control effect was calculated by the control value according to the following equation 1, inoculation and induction for each plant disease was carried out as follows.

Test for Rice Blast (RCB)

The pathogen Magnaporthe oryzae KJ201 strain was inoculated in rice bran agar medium (rice polish 20 g, dextrose 10 g, agar 15 g, distilled water 1 L) and incubated in a 25 ° C. incubator for 2 weeks. The surface of the media was grown with a sterile brush to scrape the surface of the media to remove the aerial hyphae, and spores were formed for 48 hours on a fluorescent lighted shelf (25 ± 1 ° C.). Inoculation of pathogens was made by spraying enough condensed spores to produce a certain concentration of spore suspension (1 × 10 ⁶ spores / ml) with sterile distilled water and then to flow down the plants. Inoculated rice was treated in the dark for 24 hours in a dark state, and placed in a constant temperature and humidity room with a relative humidity of 80% or more and a temperature of 25 ± 1 ° C. for 5 days, and then the leaf area ratio of the leaves was examined.

시험 Test for Rice Sheath Blight (RSB)

After the appropriate amount of bran in a 1 L culture bottle was sterilized and inoculated with Rhizoctonia solani AG1 bacteria and incubated in a 25 ℃ incubator for 7 days. Pathogen inoculation was inoculated evenly in the pot where rice was grown by finely crushing the cultured mycelial mass, incubated for 3 days in a humidified phase (25 ℃), and then placed for 4 days in a constant temperature / humidity chamber having a relative humidity of 80% or more to investigate the disease occurrence. . The incidence survey was to investigate the lesion area rate of rice leaf sheaths.

시험 Test for Cucumber Gray Mold (CGM)

Botrytis cinerea NY76, an asymptomatic fungal pathogen, was inoculated in potato agar medium for 5 days at 20 ° C thermostat (cancer), and then cultured for another 7 days while crossing light cancer for 12 hours a day. Was used as the inoculum. Spores formed on the medium were harvested using a potato liquid medium, and the spore concentration was adjusted to 5 × 10 ⁵ spores / ml using a hemocytometer, and then sprayed with one leaf cucumber seedlings. Cucumber seedlings were inoculated for 3 days at 20 ° C (95% relative humidity) and examined for the area area of cucumber leaves.

시험 Test for tomato late blight (TLB)

The pathogen Phytophthora infestans PIT strain was inoculated in oatmeal agar medium and incubated in a 20 ° C thermostat. The spermatozoa formed in the culture medium was harvested by adding sterile distilled water, and spores concentration was measured by using a hemocytometer under an optical microscope to make a spore suspension of 3 × 10 ⁴ sporangia / ml. . The 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.

시험 Test for Wheat Leaf Rust (WLR)

The pathogen, Puccinia recondita , is a live parasite, and as a result, the spores formed in the wheat seedlings were used as inoculum while the plants were passaged directly to plants. Spore suspension (spore 0.67 g / L) was prepared from harvested spores, and then sprayed with wheat seedlings. The seedlings inoculated 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 the inoculation.

시험 Test for Barley Powdery Mildew (BPM)

Since the pathogen Blumeria graminis f. Sp. Hordei is a biotic parasitic bacterium, powdery mildew spores formed on barley leaves artificially inoculated from barley seedlings were used as the inoculum. The inoculated barley seedlings were inoculated evenly and inoculated. The inoculated barley seedlings were placed in a constant temperature and humidity room at 20 ° C. and a relative humidity of about 70% for 7 days, and then the lesion area ratio was examined.

The incidence of untreated control was 30% for rice blast, 95% for rice leaf blight, 53% for cucumber gray mold, 70% for tomato late blight, 70% for wheat blight, 15% for wheat rust, and 30% for barley flour. In all, there was sufficient disease development to assess the efficacy of the KRICT3 strain. As can be seen in Table 5, when treated with the spore suspension of S. simulilium lamelicola KRICT3 strain excellent control over 90% against rice leaf blight, cucumber ash mold, tomato blight and barley powdery mild disease except wheat rust disease It was confirmed that the effect was shown, and showed 80% control effect against rice blast. When the culture filtrate of S. simillium lamelicola KRICT3 strain was sprayed, it was confirmed that it has 72% and 71% control effect against cucumber gray mold and barley powdery mildew, respectively. It was confirmed that it produces antibacterial substances that can suppress the occurrence of gray mold and powdery mildew.

From the above results, it could be confirmed that the Simplicillium lamelicola KRICT3 strain has an excellent effect on the control of various plant diseases, that is, rice blast, rice leaf blight, cucumber ash fungus, tomato blight, and barley flour.

2) Simplicillium Ramelicola KRICT3  Effect of Spore Concentration on Strains of Tomato Blight

Simplicirium lamelicola KRICT3 strains were inoculated into potato liquid medium and shaken at 150 rpm for 10 days at 20 ° C. The simplicity lamellicola KRICT3 culture solution was filtered with a two-ply gauze to remove the cells and centrifuged to remove the spore suspension harvested with sterile distilled water, and then each 1 × 10 ⁴ , 1 × 10 ⁵ , 1 × 10 ⁶ , A spore suspension of 1 × 10 ⁷ and 1 × 10 ⁸ spore concentrations was prepared.

Two to three leaf tomato seedlings grown in a greenhouse and adult tomato plants grown for about two months were sprayed with spore suspension of S. similrium lamelicola KRICT3 strain, and then 1 day afterward, gray mold (spore concentration: 1 × 10 ⁶ spores / ㎖) inoculation and induction of the disease after the investigation was carried out to investigate the gray mold control effect is shown in Table 6 below. The incidence of untreated control was 89% in seedling tomato asymptomatic disease and 40% in adult tomato asymptomatic disease.

As can be seen from the results of Table 6 above, the treatment of S. similrium lamelicola KRICT3 strain at a concentration of at least 1 × 10 ⁷ spores per ml was able to control more than 90% of gray mold disease in both seedlings and adult plant tomatoes. When KRICT3 strain was treated at a concentration of 1 × 10 ⁵ to 1 × 10 ⁶ spores per ml, it was confirmed that gray mold disease can be inhibited by 27% to 62% compared to the untreated group.

3) Effect of Controlling Lily Leaf Blight and Red Pepper Plague on Culture Medium Concentration

Simplicirium lamelicola KRICT3 strains were inoculated in potato liquid medium and shaken at 150 rpm for 10 days at 20 ° C. The simplicity lamella cola KRICT3 culture solution was filtered with a two-ply gauze to remove the cells, diluted three times each with sterile distilled water to prepare a three-fold, nine-fold, 27-fold, 81-fold, 243-fold dilution, respectively, of the stock solution Spore concentration was about 1 × 10 ⁸ per ml.

In the experiment of lily ash fungus, the lily plants were planted in the gardening soil and sprayed with a culture solution of the strain of Simlyillium lamelicola KRICT3 on the plant just before flowering. Botrytis elliptica , spore concentration: 5 × 10 ⁵ spores / ml), and after induction of disease, the disease was investigated to investigate the effect of leaf blight control.

The pepper plague experiment was carried out by irrigating the culture solution of S. simillirium lamelicola KRICT3 strain on the pepper plants of the primary branch planted in gardening soil for 2 months in sowing pepper seeds (varieties: Bugang, Monsanto Korea). After 1 day, the inoculation of pepper blight bacteria (pathogen: Phytophthora capsici CY6225, spore concentration: 1 × 10 ⁴ sporangia / mL) was inoculated and the disease was induced. The incidence of untreated control was 50% for lily leaf blight and 38% for pepper blight, indicating that there was enough plant disease to evaluate the efficacy of the KRICT3 strain.

As can be seen in Table 7, the Simlyillium lamella cola KRICT3 strain was diluted 243 times (4.1 × 10 ⁵ spores / ml) of the culture cultured at 20 ° C. for 10 days as a control against lily gray mold. Even when sprayed with, it was confirmed that exhibited a high control effect of 95%. In addition, the control effect against the pepper blight of the Simlyillium lamelicola KRICT3 strain was somewhat lower than that of lily leaf blight, but a 27-fold (3.6 × 10 ⁶ spores / ml) diluent showed a high control effect of 79%. I could confirm it.

4) Simplicillium Ramelicola KRICT3  Sustained drug efficacy

Simplicirium lamelicola KRICT3 strains were inoculated in potato liquid medium and shaken at 150 rpm for 10 days at 20 ° C. Filter the culture medium of the Simplicrium lamelicola KRICT3 strain with a two-ply gauze to remove the cells and dilute with sterile distilled water to prepare a spore suspension of KRICT3 strain of 1 × 10 ⁷ spores / ml and 1 × 10 ⁸ spores / ml, respectively. It was. Tomato plants grown in a greenhouse at 20 ± 5 ° C. for about 2 months were inoculated with spore suspensions of two concentrations of S. similrium lamelicola KRICT3 strain, respectively, 1, 2, 3 and 5 days. Sprayed 7 days ago and grown in a greenhouse, followed by gray mold (pathogen: Botrytis cinerea , spore concentration: 5 × 10 ⁵ spores / ml) and inoculated disease was investigated after the disease was investigated to investigate the effect of gray mold disease. The incidence of untreated control was more than 50%, indicating that there was sufficient tomato gray mold disease to evaluate the efficacy of KRICT3 strain.

As can be seen in Table 8 above, even after inoculated with the gray mold fungus after incubation for 7 days in a concentration of 1 × 10 ⁸ spores / ㎖ simplicity lamella cola KRICT3 strain in a greenhouse It was confirmed that control could be performed.

In addition, when treated with 1 × 10 ⁸ spores / ml concentration of the Simlyillium lamelicola KRICT3 strain, the control effect was 92% or more until 3 days after the treatment and 78% after 5 days. It was. Therefore, it could be confirmed that the Simpliclium lamelicola KRICT3 strain survived on the surface of the plant for a long time even after spraying it on tomato plants to control gray mold disease.

Korea Biotechnology Research Institute KCTC11765BP 20100915

<110> KOREA RESEARCH INSTITUTE OF CHEMICAL TECHNOLOGY <120> Simplicillium lamellicola KRICT3 strain, composition for control          plant diseases and control method of plant diseases with same <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 774 <212> DNA <213> Simplicillium lamellicola <220> <221> gene (222) (1) .. (774) <223> 28S rDNA <400> 1 ttgccccagt aacggcgagt gaagcggcaa cagctcaaat ttgaaatctg gctcctagag 60 tccgaattgt aatttgcaga ggatgctttt gatgcggtgc cttccgagtt ccctggaacg 120 ggacgccata gagggtgaga gccccgtctg gtcggatgcc aaatctctgt aaagctcctt 180 cgacgagtcg agtagtttgg gaatgctgct ctaaatggga ggtatatgtc ttctaaagct 240 aaatattggc cagagaccga tagcgcacaa gtagagtgat cgaaagatga aaagcacttt 300 gaaaagaggg ttaaaaagta cgtgaaattg ttgaaaggga agcgcctatg accagacttg 360 ggcccggtga atcatccagc gttctcgctg gtgcactttg ccgggcacag gccagcatca 420 gtttgccttg ggggaaaaag gctttgggaa tgtagctcct tcgggagtgt tatagaccat 480 tgcataatac cctggggcgg actgaggttc gcgcatctgc aaggatgctg gcgtaatggt 540 catcagcgac ccgtcttgaa acacggacca aggagtcgtc ttcgtatgcg agtgttcggg 600 tgtaaaaccc ctacgcgtaa tgaaagtgaa cgcaggtgag agcttcggcg catcatcgac 660 cgatcctgat gttctcggat ggatttgagt aagagcatac ggggccggac ccgaaagaag 720 gtgaactatt cctgtatagg gtgaagccag aggaaactct ggtggaggct cgca 774 <210> 2 <211> 551 <212> DNA <213> Simplicillium lamellicola <220> <221> gene (222) (1) .. (551) <223> ITS region <400> 2 attaacgagt tatacaactc ccaaaccctt tgtgaacctt accacttacg ttgcttcggc 60 ggaacgcgct ggtgtcgccc tcacgggtgc cccaggtcaa cgcgcccgcc ggggactaca 120 aactcttgat tttgcgaaag cagtattctt ctgagtggcc gaaaggcaaa aaaacaaatg 180 aatcaaaact ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcaaaat 240 gcgataagta atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc 300 gcccgccagc attctggcgg gcatgcctgt tcgagcgtca tttcaaccct cgacttcacg 360 tctttacgga cgagaagccg gtgttggggc gcggcgatcg agcctagctc gcgccggctc 420 cgaaatttag tggcggcccg ttgcggcgac ctctgcgtag taacttaacc tcgcactggg 480 acagcagcgc ggccacgccg taaaaccccc gactttttta aggttgacct cgaatcaggt 540 aggactaccc g 551

Claims (6)

Florisil simple Solarium Melilla la cola (Simplicillium lamellicola ) KRICT3 strain (KCTC 11765BP). Simplicillium lamellicola KRICT3 strain (KCTC 11765BP), rice blast, rice leaf blight, tomato blight, pepper blight, cucumber gray mold, tomato gray mold A composition for controlling at least one plant disease selected from the group consisting of bottles, lily leaf blight and barley powdery mildew. delete The method of claim 2,
The plant diseases include Alternaria alternata , Cochliobolus heterostrophus , Magnaporthe oryzae , Botrytis cinerea , Botrytis ali ( Botrytis allii ), Sclerotinia sclerotiorum , Colletotrichum orbiculare , Phytophthora capsici , Phytophthora infestans , Fusarium oxysporum , Pythium ultimum , Pythium graminicola and Rhizoctonia solani ) A composition for controlling plant diseases, which occurs by AG1 or the like. The method of controlling plant diseases comprising spraying the composition for controlling plant diseases of claim 2 on soil, leaves, stems, flowers or fruits of crops. 6. The method of claim 5,
The method of claim 1, characterized in that the simplicity lamellicola KRICT3 strain is treated in an amount of 1 × 10 ⁴ to 1 × 10 ⁹ spores / ml.

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