KR101791569B1 - microorganism agent for biocontrol plant diseases using Trichoderma virens TR1047 and Trichoderma harzianum TR1048 - Google Patents

Abstract

The present invention aims to provide a new microorganism strain TRICO DELMARBIRES TR1047 and TRICODELMADHANUM TR1048 which have a dragging power against plant scleroderma and black rot mycorrhiza. Especially, the present invention relates to a microorganism which is resistant to scleroderma or black decay scleroderma It has a much better pathway resistance and is intended to provide a new microorganism strain that can replace chemical pesticides by itself.

Description

[0001] The present invention relates to a microorganism for microbial control using Trichoderma virens TR1047 and Trichoderma harzianum TR1048,

The present invention relates to the control of sclerotia-related diseases, in particular sclerotinia and black rot mycotic disease, and more particularly to a microorganism comprising a natural tricortalma, a separated trichoderma, a method for producing such a microorganism, .

Sclerotium is a term derived from Greek skleros (hard). It is a rigid sphere formed by dense aggregation of hyphae. It does not form spores on the inside or on the inside with a clumpy mass, and fruiting bodies are often formed on sclerotia. Although many fungi form sclerotia, Claviceps parasitic on parasitic plants The sclerotia formed by the spp . contains alkaloids of erythrocytes, and the inclusion of sclerotia is a problem in imported grains. In crops, especially vegetables, disease caused by sclerotinia occurs well in the pavement, and it is characterized by black sclerotia when the onset becomes severe. In some parts of Aspergillus , sclerotia is formed, and A. flavus , A. ochraceus Are often formed on the medium. It is a durable tissue with a long durability. It survives the bad conditions such as dry soil, and forms fruiting bodies and spermatogonial structures under hostile conditions to disperse spores (see Food Science and Technology Encyclopedia).

Sclerotia has been a cause of human health problems from ancient times, in the Middle Ages Claviceps Eating roasted bread with rye infected with purpurea sclerotia and ergot poisoning caused thousands of people to die or become deformed (1). The ergot is known to have been used as a medicament since it has a strong uterine contraction function, promotion of labor and hemostatic function at the time of delivery. There are 14 known isomers of seven pairs of isomeric acid, isomeric acid and its stereoisomer, ricerugic acid , Ricergic acid is the parent of the physiological action is strong, but the stereoisomer is weak physiological function.

Sclerotia occurs in various crops and causes damage to agriculture. Sclerotinia Sclerotinia is caused by lettuce by sclerotiorum and S. minor . The lettuce sclerotia was reported in the USA in 1890 (2), and it was first reported in 1976 in Korea (3). In the United States and Europe, S. sclerotiorum is mainly infected by ascospores (4). In major countries producing lettuce such as the United States and Europe, lettuce sclerotinia is known to be an important disease causing loss of yield. In the United States, 175% It is known as a disease causing crop loss (5). Lettuce, which is produced all year round in Korea, is mainly cultivated in the facility, and the environment in the cold and humid environment during the winter from late autumn to early spring promotes the occurrence of scleroderma, thereby causing economic damage to farmers.

Black rot mycotic disease, which occurs during the cultivation of zephyrum and garlic, is known to be one of the important plant diseases threatening stable production. The incidence of black rot mycotic disease has been reported in several regions. In the UK, 10% of the production was reduced to black rotten sclerotinia, and in Mexico and Brazil, harvesting was not possible at all (7). In Canada, the incidence of the disease in the packaging was 65% (8), and in Ethiopia in 2003 and 2004, 96.6% of the packaged products had black rot mycosis (9). In Korea, since the first reported in Goheung in 1988, lilies and vegetables have been growing up to 1996 (10). However, according to Kim (2001), the incidence of onset of black rot in 2000 was 44.7% and the incidence rate of incidence was 19.6% in 2000. However, the incidence rate of incidence in 2001 and 2003 was 20.4% and 14.3% And 10.1%, respectively (11, 12).

In order to control scleroderma, Trichoderma biological methods using microorganisms such as harzianum and Bacillus subtilis , physical methods of disinfecting the soil with solar heat, and chemical methods of treating bactericides have been used (7, 13). Although many microorganisms such as Trichoderma , Bacillus, and Burkholderia have been used to obtain excellent control effects, they have been shown to have low efficacy depending on the package or not meet the expectations (14, 15, 16). Solar disinfection, which reduces the viability of sclerotia by increasing the temperature of the soil by covering the package with a plastic film, showed excellent control effect against black decaying sclerotia in packaging (17, 13). However, when 1 to 10 sclerotides are present in 1 g of soil, the incidence rate reaches 100% in the package and 0.001 in case the disease occurs in a small number of plants, so that the density of the pathogens present in the soil is reduced to a very low level It is difficult to obtain excellent effects continuously (18, 7). Kim et al. (19) used seedling sowing prior to mid-October or sowing seedlings at deep seeding to reduce disease incidence. In addition, lime was applied to the soil to inhibit disease development, but it was not as good as when treated with a disinfectant. The use of disinfectants is known to be the most common method, and various disinfectants such as dicloran, iprodione, procymidone, and tebuconazole have been used for disease control. However, since the treated disinfectant is decomposed in the soil or can not be controlled by the treatment once, it can be satisfactorily effected by various treatment methods or periodically continuous treatment or by varying the amount depending on the density of cultivated crops (20, 21, 9). Despite the reports of various control methods, it is necessary to understand physiological and ecological characteristics of pathogens and disease in order to control sclerotia. In order to control sclerotinia both domestically and externally, various pesticide-based control studies have been conducted. However, as the pesticide residue problem and the demand for environmentally friendly agricultural products have increased, various methods to replace chemical control have been studied.

Chemical scavengers are the most commonly used to control scleroderma or black decay scleroderma. However, chemical pesticides cause acidification and contamination of the soil, toxicity to the drawing, and environmental problems caused by residual pesticides. Recently, due to environmental awareness raising and consumers' preference for eco-friendly food, it is a tendency to refrain from the use of chemical pesticides and to increase the use of eco-friendly farm materials instead. As environmentally friendly farming materials, natural pesticides extracted from plants and biological pesticides using microorganisms are mainly used. Although the interest and use of environmentally friendly farming materials are gradually expanding, it is still inadequate to replace existing chemical pesticides.

Open No. 10-2009-0063987 discloses a novel Streptomyces sp. Strain having an antimicrobial activity against a plant pathogenic bacterium that causes gray mold, gray mold rot, black spotted bottle, red pepper anthracnose, And a plant pathogenic bacterium. In patent application No. 10-2011-0055458, a novel microcystic fungus having an antifungal activity, Xylogone ganodermophthora ) and a composition for controlling plant diseases, which comprises the same as an active ingredient. In Patent Application No. 10-2010-0116562, antagonistic action against rice sheath blight, tomato gray mold, tomato plague, wheat red rust, barley powdery mildew, red pepper anthracnose, tomato foot rot, tomato wilt disease and tomato root blight nematode Bacillus velezensis strain G341 and a plant disease control method using the same. However, since most of the biological pesticides can not completely replace the chemical pesticides in the single use, there is a demand for the development of the pesticides having a level of control agent that can replace chemical pesticides .

The present invention has found that TRICO DELMARBIRES TR1047 and TRICO DELMHAMENUM TR1048 isolated from soil in Korea have excellent effect in controlling scleroderma, and found that TRICO DELMARBIRES TR1047 and TRICO DELAMAS The inventors have completed the present invention by producing plant microbial control microorganism using Hyalinum TR1048.

(1) L. R. TULASNE, Annales des Sciences Naturelles, Botanique, 3rd series, vol.20, pp.5-56 (1853)

(2) SUBBARAO, Plant Dis., Vol. 82, pp. 1068-1078 (1998)

(3) KIM K. C., Korean J. Plant Protect, vol. 15. pp. 223-243 (1976)

(4) Abawi, G. S. and Grogan, R. G., Phytopathology, vol. 65, pp. 300-309 (1975)

(5) Purdy, L. H. Phytopathology 69: 875-880 (1979)

(6) Chang, S. W. and Kim, S. K., Plant Pathology J., vol. 19, pp. 79-84 (2003)

(7) Entwistle, A. R., Rabinowitch, H. D. and Brewster, J. L. Eds: CRC Press, Boca Raton, USA. pp. 103-154 (1990)

(8) Earnshaw, D., M. R. McDonald and G. J. Boland, Can. J. Plant Pathol. vol. 22, pp. 387-391 (2000)

(9) Zewde, T., C. Fininsa, P. K. Sakhuja and S. Ahmed, Crop Prot., Vol. 26, pp. 1566-1573 (2007)

(10) Cho W. D. and W. G. Kim, J. Plant Pathol., Vol. 12, pp. 251-254 (1996)

(11) Kim, C. H., Res. Plant Dis., Vol. 8, pp.1-10 (2002)

(12) Kim, C. H., Res. Plant Dis. vol. 10, pp. 1-7 (2004)

(13) Melero, J. M., R. Gonzalez, J. Gomez, J. Bejarano and M. J. Basallote, Plasticuiture, vol. 82, pp. 73-82 (1989)

(14) Han, K. S., B. R. Kim, S. T. Kim, S. S. Hahm, K. H. Hong, C. K. Chung, Y. G. Nam, S. H. Yu and Jae Eul Choi, Res. Plant Dis., Vol. 19, pp. 21-24 (2013)

(15) Lee, S. Y., S. B. Lee, Y. K. Kim and S. J. Hwang, Res. Plant Dis., Vol. 12, pp. 81-84 (2006)

(16) Melero-Vara, J. M., A. M. Prados-Ligero and M. J. Basallote-Ureba, European J. Plant Pathol., Vol. 106, pp. 581-588 (2000)

(17) Satour, M. M., M. F. Abdel-Rahim, T. Yamani, A. Grinstein, H. D. Rabinowitch and J. Katan, Acta Hort. vol. 255, pp. 151-159 (1989)

(18) Crowe, F. J., D. H. Hall, A. S. Greathead and K. G. Baghott, Phytopathology, vol. 70, pp. 64-69 (1980)

(19) Kim, Y. K., M. K. Kwon, H. S. Shim, T. S. Kim, W. H. Yeh, W. D. Cho, I. H. Choi, S. C. Lee, S. J. Ko, Y. H. Lee and C. J. Lee, Res. Plant Dis., Vol. 11, pp. 28-34 (2005)

(20) Groves, K. and K. S. Chough, J. Agri. Food Chem. vol.18, pp. 1127-1128 (1970)

(21) Entwistle, AR, Aspects Appl. Biol., Vol. 40, pp. 166-175 (1986)

The present invention aims to provide a new microorganism strain having a drag resistance against the sclerotinia and black rot mycorrhaphy of plants and has a far superior drag resistance than the known microorganisms of scleroderma or black decay scleroderma, And to provide a new microorganism strain capable of replacing pesticides.

The present invention also provides a microorganism preparation for controlling scleroderma, which comprises the microorganism and the microorganism, and a preparation method thereof.

Another aspect of the present invention is to provide a method of controlling microorganisms for controlling sclerotinia and black rot mycobacteria.

In the present invention, Tricodelma virans strain TR1047 and Tricodelma javanum strain TR1048, which have a dragging resistance against scleroderma and black rot mycosis of plants, are provided.

In addition, the present invention provides a microorganism preparation for controlling scleroderma and black decaying sclerotinia comprising the above-mentioned trico delmareviris TR1047 or tricodelmajalanum TR1048 and a culture solution of the strain as an active ingredient.

In addition, in the present invention, TRICODELMARBIRES TR1047 or TRICO DELMASHANUM TR1048 was cultured in a PDA medium for 15 days to prepare a spore suspension, and the concentration was adjusted to 5x10 ⁶ spores / ml through a hemocytometer. There is provided a method for controlling scleroderma and black decaying scleroderma comprising inoculating two barley cultivated with sclerotinia bacteria into a soil and locating the spore suspension per Trichoderma strain at a rate of 5 ml per 1 lettuce.

As can be seen from the examples, the composition of sclerotinia and black rot mycotic disease control agent obtained according to the present invention exhibits excellent sclerotinia and black rot mycorrhiza control activity of not less than 90% by itself, And can be used as effective sclerotinia and black decay scleroderma control agents. The sclerotinia and black rot mycotic disease control composition of the present invention has an excellent effect that can solve problems of soil contamination and toxicity to humans, and can be effectively used for sclerotinia and black rot mycorrhiza which have resistance against chemical pesticides.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow diagram of the strain of the present invention in accordance with the nucleotide sequence similarity (ITS 1 and ITS 2).
2 is a flow diagram of the strain of the present invention according to nucleotide sequence similarity (Elongation factor-1a).
Fig. 3 shows the morphological characteristics of strains of T. virens TR1047 (A) and T. harzianum TR1048 (B) obtained by optical microscopy.
Figure 4 is a graph virens TR1047 and T. harzianum TR1048, and parasites of sclerotia and hyphae of S. cepivorum .
FIG. 5 is a graph showing the infection rate of Sclerotinia sp.
Fig. 6 is a graph showing the spore germination rate by temperature and spore concentration of T. virens TR1047 strain.
Fig. 7 is a graph showing the spore germination rate by temperature and spore concentration of T. harzianum TR1048 strain.

The present invention relates to a novel Tricordel mabirence TR1047 having a controlling effect against scleroderma and black rot mycorrhage, a method for culturing and producing the same, a composition for controlling scleroderma and black decaying scleroderma using the same, and a method for the formulation and control thereof.

The present invention also relates to a method for culturing and producing TRICO DELMASHANUM TR1048, a composition for controlling scleroderma and black decaying scleroderma using the same, a method of formulating the same, and a method for controlling the same.

The TRICHL DELMARBIRES TR1047 and TRICODELMADHANUM TR1048 strains according to the present invention have an excellent controlling effect especially on the lettuce sclerotinia and the black rot rot mycorrhizal disease, and thus are useful as a pesticide for preventing plant diseases such as lettuce scurvy, .

The inventors of the present invention have completed the present invention by discovering that Tricodelma virans TR1047 and TR108 strains selected as antagonistic strains against lettuce sclerotinia and black rot rot mycorrhizae have an excellent effect on control of lettuce sclerotinia and black rot rot mycorrhizae .

First, the present invention provides novel TRICODELMARE TR1047 and TRICODELMADHANUM TR1048 strains having excellent control effects against phytopathogenic fungi. The TRICHEL DELMARBIRES TR1047 and TRICODELMADHANUM TR1048 strains were newly isolated and identified as having excellent control effects on lettuce sclerotinia and black rot rot mycorrhizae, and were given accession numbers of KACC 93157P and KACC 93158P.

In yet another aspect, the present invention provides a method of culturing TRICODEL DEL BIRRENS TR1047 and TRICODELMADHANUM TR1048.

The medium used in the culture method of the present invention is a CMD medium consisting of cornmeal agar (Difco) + 2% dextrose and 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO47H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 20 g agar, 10 mg chlorotetracycline, 50 mg dihydrostreptomycin sulphate, and 100 mg penicillin G.

In the culturing method of the present invention, the culture temperature is preferably 25 to 35, preferably 30, and the culture pH is preferably 4 to 8, preferably 5.5 to 6.5, and the incubation time is preferably 72 hours or less, More preferably 36 hours to 52 hours. The range of temperature, pH, and time is a value obtained under the optimum condition under experimental conditions.

In yet another aspect, the present invention provides a composition for controlling phytopathogenic fungi, comprising TRICO DELMARBIRES TR1047 and TRICODELMAZANUM TR1048 or a culture thereof.

The phytopathogenic fungi include Rhizoctonia solani , Fusarium oxysporum , Fusarium solani and Pythium ultimum , Phytophthora capsici , Sclerotinia sclerotiorum , Sclerotinia minor, Sclerotium cepivorum, Sclerotium sp, Botrytis cinerea . The plants to which the composition of the present invention can be applied may be any of the above microorganisms, particularly all plants in which scleroderma and black rot mycosis occur, such as lettuce, green pepper, tomato, strawberry and cabbage .

The microbial composition of the present invention may be one comprising the above-mentioned TRICODELMARAVIRES TR1047 and TRICODELMAZHANUM TR1048 strain itself, and may be a culture obtained by culturing it under a specific condition in a specific medium.

The culture may be obtained by culturing in a medium supplemented with at least one carbon source selected from the group consisting of carbon sources selected from the group consisting of molasses, barley, PDA, rice bran and the like. In the present invention, among the carbon sources, the barley was found to be effective for the mass production of TRICODELMAVIRUS TR1047 and TRICODELMAHANUM TR1048.

The composition for controlling phytopathogenic bacterium and / or fungi according to the present invention, which contains Tricodelma virans TR1047 and Tricodelma javanum TR1048 strain or a culture thereof, comprises one kind selected from the group consisting of biopolymers, Or may further be formulated including the above additives. The formulated composition is preferably a wettable powder or liquid wettable powder which can be easily applied to plants by an easy method such as spraying.

The biopolymer material is used as a delivery medium for formulation in the preparation of a liquid wettable powder of a microbial composition, and it is transparent and maintains a proper viscosity without remaining a stain. Examples thereof include corn starch, modified starch, tapioca starch, , Sunflower, sunflower, sun cream, suntan, sunshine, low-grade pregel, sun cap, sun super gel, sun batters, zeolite and the like. The content of the biopolymer in the composition of the present invention may be adjusted so that the formulated composition is transparent and maintains a proper viscosity without remaining a trace. The content of the biodegradable polymer in the composition of the present invention is 100 wt. And 30 to 50 parts by weight.

The protecting agent may be at least one kind selected from the group consisting of brown rice oil and olive oil, and its content is 2 to 10 parts by weight, preferably 2 to 10 parts by weight, per 100 parts by weight of the cultivars TRICODELMARBREX TR1047 and TR1048, Is preferably 3 to 7 parts by weight. The nutrient may be at least one selected from the group consisting of sucrose and fructose. The nutrient may be contained in an amount of 1 to 5 wt.% Based on 100 parts by weight of the cultivars TRICODELMARBIRES TR1047 and TR1048, %, Preferably 1.5 to 3.5 wt%. At this time, sucrose is a nutrient source of antagonistic antigens. It is believed that the flavor and taste of amino acid oil and olive oil are protected from ultraviolet rays by coating Tricodelma virans TR1047 and TR1048 and TR1048, and they act as an emulsifier which mixes the ingredients well.

The formulated composition of the present invention not only has a good control effect against phytopathogenic fungi but also does not contain chemicals harmful to living organisms and is very safe. Even if stored for 3 or more months at room temperature, the microorganism population is stable So that the storage stability is excellent.

In another aspect, the present invention provides a method of controlling phytopathogenic bacteria and / or fungi using the composition. In the controlling method of the present invention, the application concentration of the composition can be appropriately adjusted in consideration of the crop to be applied, the distribution of insect pests and the like, and is generally diluted to 10 to 1000 times, preferably 100 to 500 times Can be used.

The phytopathogenic fungus to which the present method of prevention is applicable is Rhizoctonia solani , Fusarium oxysporum , with Fusarium solani Pythium ultimum , Phytophthora capsici , Sclerotinia sclerotiorum , Sclerotinia minor, Sclerotium cepivorum, Sclerotium sp , Botrytis cinerea . < / RTI > The plants that can be used for the above-mentioned control method may be those selected from the group consisting of the above-mentioned bacteria, particularly all plants in which sclerotinia and black rot mycosis occur, such as lettuce, side waves, tomatoes, strawberries and cabbages.

In another aspect, the present invention provides a composition for controlling phytopathogenic fungi, which contains, as an active ingredient, Tricodelma virans TR1047 and TR108 strain selected as having a controlling effect against a plant pathogenic bacterium or a culture thereof do. The phytopathogenic fungi include Rhizoctonia solani , Fusarium oxysporum , Fusarium with solani Pythium ultimum , Phytophthora capsici , Sclerotinia sclerotiorum , Sclerotinia minor, Sclerotium cepivorum , Sclerotium sp , Botrytis cinerea . < / RTI >

The culture may be obtained by culturing Trichoderma virans strain TR1047 and TR10848 strain in a CMA or SNA medium. It may also be obtained by mass-culturing in molasses, barley, PDA or rice bran.

The composition containing the TRICODEL DEL BIRRENS TR1047 and TRICODELMADHANUM TR1048 or a culture thereof may further contain at least one additive selected from the group consisting of a biopolymer, a protective agent and a nutrient. The biopolymer material may be selected from the group consisting of corn starch, modified starch, tapioca starch, meju powder, rice bran, sun cream, suntaner, sunsize, low-pregel, sun cap, sun super gel, sun batters, At least one selected from the group consisting of sucrose and fructose, and the protective agent is at least one selected from the group consisting of brown rice oil and olive oil, and the nutrient may be at least one selected from the group consisting of sucrose and fructose.

In a preferred embodiment, the composition comprises 30-50 parts by weight of tapioca starch, 2-10 parts by weight of olive oil, and 10-40 parts by weight of a culture of a strain of Trichoderma virans strain TR1047 and Trichoderma japonicum TR1048 and 100 parts by weight of the culture, And 1 to 5 parts by weight of sucrose.

In another aspect, the present invention relates to a method for controlling phytopathogenic fungi, which comprises using the above-mentioned Tricodelma virans TR1047 and TR1048 strain or a culture thereof as an active ingredient, Rhizoctonia solani , Fusarium oxysporum , Fusarium with solani Pythium ultimum , Phytophthora capsici , Sclerotinia sclerotiorum , Sclerotinia minor, Sclerotium cepivorum , Sclerotium sp, Botrytis cinerea . < / RTI > The plants which can be used for the above-mentioned control method may be any of the above-mentioned fungi, particularly all plants in which scleroderma and / or black rot mycosis occurs, such as lettuce, side waves, tomatoes, strawberries and cabbages .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1

Molecular identification of the strain

 DNA of two strains was extracted according to the method of Edwards (1991). Trichoderma mycelia cultured on PDA medium for 3 days were plated out with platinum, put in microtube (2 ml), lyophilized for 24 hours, and homogenized. Lysis buffer 500 was added, mixed and reacted for 1 hour at 65 ° C. Then, CTAB 500 was added to react at 65 for 15 minutes. After the addition of PCI (phenol / chloroform / isoamylalcohol = 25: 24: 1), the supernatant was transferred to a new microtube. Isopropanol was added to precipitate the DNA, followed by washing with 1 ml of 70% ethanol and drying the pellet. The pellet was dissolved in 100 TE buffer (10 mM Tris HCl - pH 8.0, 1 mM EDTA - pH 8.0), and Rnase (10 mg / ml) 2 was added and reacted at 60 for 30 min. The extracted DNA was stored at -70 and used for the experiment.

  PCR amplification and sequencing were performed using ITS1 (5'-TCCGTAGGTGAACCGCGG-3 ') and ITS2 (5'-TCCTCCGC TTATTGATATGC-3') primers in the ITS1 and ITS2 regions on rDNA (White et al., 1990) EF1-728F (5'-CATCGAGAA GTTCGAGAAGG-3 ') and EF1-986R (5'-TACTTGAAGGAACCCTTAC-3') primers were used in the factor-1a gene region (Druzhinina et al., 2004). PCR was carried out by adding 100 ng of genomic DNA, 10 pM of primer, 0.25 of Tag DNA polymerase, 0.2 mM dNTP 1, 10 x buffer 2, and final volume of 50 to sterilized water. PCR was carried out by denaturation at 94 for 4 min, followed by denaturation at 94 for 1 min, annealing at 56 for 1 min, and extension at 72 for 2 min. The PCR product was electrophoresed on 1.5% agarose gel and stained with ethidium bromide. A 100 bp DNA ladder was used as a marker. Sequencing was performed using the PROMEGA PCR Clean-up System kit, followed by BLAST searches of the National Center for Biotechnology Information (NCBI) for comparison and analysis with existing DNA sequences. (Figs. 1 and 2)

T. virens , T. asperellum , TR1048, TR1050, and TR1085 strains were identified as T. harzianum in the TR1047 and TR1115 strains, respectively, in the ITS region and elongation factor-1a region of 5 selected strains. (Figs. 1 and 2)

Example 2

Investigation of morphological characteristics of strains

 TR1047 and TR1048 were cultured in CMD (corn meal agar + 2% dextrose, Difco) medium at 20 for 2 days. The sterilized slide glass was immersed in 50 water agar medium, coated, and placed on agar plate medium to prepare a plate. Five strains of mycelia were transplanted onto the slide glass of the prepared plate and cultured for 25 days. The shape and size of the sporangia and spores were examined under the light microscope.

The results are shown in Fig.

Example 3

Investigation of culture characteristics of strain

end. Incubation temperature

In order to investigate mycelial growth according to the culture temperatures (5, 10, 15, 20, 25, 28, 30, 32, 35, 38, 40) of the two selected strains, Trichoderma hyphae were suspended in 20 ml of sterilized water, 200 cells were plated on PDA medium and cultured for 25 days. The cultured Trichoderma fungus was detached with a 5 mm diameter cork borer and dentated in the PDA medium. After culturing for 48 hours at each temperature, the diameter of the starting strain was examined.

Strain Pore diameter by temperature (mm) 5 10 15 20 25 28 30 32 35 38 40 TR1047 5.8 12.7 22.6 62.3 74.8 77.9 82.2 61.0 28.8 8.5 6.3 TR1048 9.4 18.2 35.1 78.2 85.0 85.0 85.0 74.7 37.9 10.8 6.5

48 hours culture, PDA medium

I. pH

In order to investigate the mycelial growth of the selected strains according to pH (3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0), Trichoderma spores were added to 20 ml of sterilized water, The cells were cultured on a PDA medium at 200 rpm for 25 days. The cultured Trichoderma fungus was detached with a 5 mm diameter cork borer and placed in the PDA medium. After culturing for 25 to 40 hours, the diameter of the selected strain was examined.

Strain Diameter of bacteria by pH (mm) 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 TR1047 50.5 54.1 58.0 58.5 59.6 61.3 58.4 58.1 56.6 55.7 53.7 51.7 TR1048 55.8 56.1 60.7 61.4 62.1 60.0 58.6 56.9 56.5 55.4 49.0 47.4

25 degrees, after 40 hours using the PDA medium, the diameters of the culture media (mm).

 All. Badge type

In order to investigate the mycelial growth and spore formation according to the selected strains, CMD medium was cultured at 20 for 2 days. The cultured Trichoderma fungus was detached with a 5 mm diameter Cork borer and dipped in the medium of PDA, CMD, MEA (malt extract agar, Difco) and SNA (Nirenberg, 1976) 35). The experiment was repeated 4 times and the diameter of the microflora was examined after 48 hours of incubation.

Strain Badge type Pore diameter by temperature (mm) 15 20 25 30 35 TR1047 PDA 24.1 66.1 80.0 85.0 48.1 CMD 30.4 76.5 85.0 85.0 60.2 MEA 28.2 67.6 83.2 85.0 49.2 SNA 29.2 78.8 85.0 85.0 49.0 TR1048 PDA 32.1 72.2 83.9 85.0 56.4 CMD 34.8 79.9 85.0 85.0 62.4 MEA 31.4 71.0 81.0 85.0 45.4 SNA 28.1 68.5 82.2 85.0 45.5

Badge type Composition (l) PDA (Potato Dextrose Agar, Difco) Potato 200g, dextrose 20g CMD cornmeal agar (Difco) + 2% dextrose MEA (Malt Extreat Agar, Difco) Malt extract 20g, Peptone 1g, Glucose 20g SNA 1 g KH ₂ PO ₄ , 1 g KNO ₃ , 0.5 g MgSO ₄ H ₂ O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 20 g agar, 10 mg chlorotetracycline, 50 mg dihydrostreptomycinsulphate, 100 mg penicillin G,

Example 4

On plant pathogens  Impact

end. Mycelial growth inhibition and Sclerotization  Research

A soil-infectious pathogen Rhizoctonia against Trichoderma sp. solani , Fusarium oxysporum , Fusarium solani and Pythium ultimum , Phytophthora The capsici was incubated in 25 thermostats, and Sclerotinia sclerotiorum , Sclerotinia minor, Sclerotium cepivorum , Sclerotium sp , Botrytis Cinerea was cultured in a thermostat for 20 days in a PDA medium, which had been placed in a 88 mm diameter Petri dish, and cultured for 20 days to investigate mycelial growth and sclerotial formation.

I. Sclerogenesis  black

Sclerotinia sclerotiorum were cultivated in barley medium for 20 to 15 days. Sclerotiae were collected and stored for 5 to 10 days. Spore suspension of the isolate was prepared. Sclerotia treated 20 sec after immersion of sclerotin was suspended in WA (water agar medium) , And were kept in a 20 ° C incubator for 3 repetitions. The incidence of sclerotia infection was examined by dissecting with a dissecting microscope and a knife.

Soil pathogen Trichoderma genus
Mycelial growth inhibition by strain Trichoderma genus
Inhibition of sclerotial formation by strain Trichoderma genus
Parasitic TR1047 TR1048 TR1047 TR1048 TR1047 TR1048 Rhizoctonia solani - - Fusarium oxysporum - - - - Fusarium solani - - - - Pythium ultimum - - - - Phytophthora capsici - - - - Sclerotium cepivorum Botrytis cinerea Sclerotinia sclerotiorum Sclerotinia  minor Sclerotium rolfsii - -

Trichoderma Investigation of the effect of bacterium on the growth of plant pathogens

: Inhibition or parasitism.

Example 5

Of the starting strain Sclerotinia Sclerotinia sclerotiorum )of  By scintillation temperature Parasitic power  black

The two selected strains were cultured in PDA medium for 25 days at 15 days, and spore suspension was prepared and then adjusted to a concentration of 5 × 10 6 spores / ml through a hemocytometer. Sclerotia of S. sclerotiorum was immersed in spore suspensions of each of the selected strains for 30 seconds and then cultured on water agar medium at 10, 15, 20, 25, 28, 30 days. The experiment was repeated 4 times and the condition of sclerotia was examined after 30 days.

The results are shown in Fig.

Example 6

Investigation of spore germination rate by temperature of strain

Spore suspension of Trichoderma 2 strain was diluted to ⁵ × 10 ⁵ /, ¹ × 10 ⁶ /, and 5 × 10 ⁶ /, and 0.25 ml / petri dish was added to water agar medium and sprout germination rate was measured after 14 hours incubation at temperature.

The results are shown in FIG. 6 and FIG.

Example 7

Investigation of light effect

To investigate the optical type mycelial growth of the Trichoderma strain 2 were removed for 2 days culture the Trichoderma colony on PDA medium with a diameter of 5mm to see cork (Cork borer). The diameter of the selected strain was examined after incubation for 25 to 40 hours with wrapping with aluminum foil.

Treatment type Pore diameter (mm) TR1047 TR1048 Cancer treatment 18.7a 24.2 a Fluorescent lamp 15.3 b 19.1 b NUV 18.0a 23.7 a

5% level by DMRT.

Example 8

Trichoderma Bacterium  Development of mass culture method

For the mass culture of TR1047 and TR1048 by using various byproducts, 5 mm diameter microflora cultivated in PDA medium was examined on the plate culture media of each kind after the fermentation for 25 to 72 hours after the fermentation.

Badge type Pore diameter (mm) TR1047 TR1048 molasses 52.77 55.27 barley 62.63 68.77 PDA 49.87 67.33 Rice bran 39.83 45.50

Example 9

Cellulase  Investigation of creation

Cells were incubated at 20 ° C for 5 min with a 5 mM culture medium grown on PDA medium. Cells were cultured at 20 ° C. Cells were stained with 0.5% congo red for 30 min and rinsed thoroughly with 1 M NaCl solution. Respectively.

Cellulase- containing medium CM-cellulose ............................................... ..... 5.0g
Tryptone ................................................. .......... 5.0g
Yeast extract ................................................ ... 5.0g
CaCl2 ................................................. ............... 1.0 g
Agar ................................................. ................ 20.0g

Medium composition

Whether to create transparency TR1047 TR1048 No treatment + + -

Example 10

Trichoderma  Strain Plant disease  Control Effectiveness

(One) Isolate  Effect of controlling the control of lettuce sclerotinia

The experiment was conducted when the third leaf stage was developed after 30 weeks' sowing in a 50 - tray tray pot mixed with a 1: 1 ratio of the lettuce and lettuce seeds. Sclerotinia were cultivated in barley medium for 40 days to form sclerotia. Excellent parasitic power against sclerotia of S. sclerotiorum Trichoderma 7 strain was cultured in PDA medium for 15 days to prepare a spore suspension, and then adjusted to a concentration of 5 × 10 ⁶ spores / ml through a hemocytometer. Then, the inoculated barley Al 2 gyunhaekbyeong culturing bacteria per 1 lettuce in soil and the spore suspension was 5ml per infusion by giving 1 lettuce by Trichoderma strains. The incidence of scleroderma was investigated at 10 days after treatment.

Strain Lee Byungju (%) Control (%) TR1047 2.2 95.5 TR1048 3.3 93.8 TR1006 21.1 60.4 TR1009 33.3 37.5 TR1012 48.9 8.3 TR1020 36.7 31.1 TR1028 11.1 79.2 No treatment 53.3 -

Effect of isolated strains on the control of scleroderma

(2) Of the starting strain  Effect of controlling the control of lettuce sclerotinia

The experiment was conducted when the third leaf stage was developed after 30 weeks' sowing in a 50 - tray tray pot mixed with a 1: 1 ratio of the lettuce and lettuce seeds. Sclerotinia were cultivated in barley medium for 40 days to form sclerotia. Superior control effect of lettuce sclerotinia 2 Trichoderma strains was made after adjusting the spore suspension and cultured 15 days in a PDA medium through hemo cytometer to 1x10 ⁶ spores / ml and 5x10 ⁶ spores / ml concentration. Then, the inoculated barley Al 2 gyunhaekbyeong culturing bacteria per 1 lettuce in soil and the spore suspension was 5ml per infusion by giving 1 lettuce by Trichoderma strains. The incidence of scleroderma was investigated at 12 days after treatment.

Treatment concentration by strain Lee Byungju (%) Control (%) TR1047 -5.0x10 ⁶ 14.4 85.3 TR1047 -1.0x10 ⁶ 28.9 70.5 TR1048 -5.0x10 ⁶ 6.7 93.2 TR1048 -1.0x10 ⁶ 37.8 61.4 No treatment 97.8 -

Effect of selective strains on the control of scleroderma

  (2) Black decay scleroderma  Investigation of the inhibitory effect

The Trichoderma 2 strain was used as a prototype. The area per treatment was 2.4 m 2, and all treatments were sown in three replicates. The Trichoderma strain was inoculated after the mass cultured T. virens TR1047 (7.5 × 10 ⁸ cfu / g) and T. harzianum TR1048 (8.4 × 10 ⁸ cfu / g) . As a control, the chemical drug Florenconazole wettable powder was sowed by treatment with 2 g / kg (side wave). The quantity of pretreatment waters was investigated except for the sickly sappan which had no commerciality.

Treated strain Lee Byungju (%) Control (%) In building
(Kg) Quantity Index (%) TR1047 7.9 51.5 4.383.3 109.4 TR1048 7.4 54.6 5.240.0 130.8 Fluroquinazol hydrate 1.4 91.4 6.423.3 160.3 No treatment 16.3 - 4.006.7 -

Trichoderma Mycelium  Treated side wave Black decay scleroderma  And In building

Example 11

Survey on the effect of crop growth promotion

   Tomato seedlings and red pepper seeds were sown on a 50-tray tray, and spore suspension (5 × 10 6 / ml) of Tr1047 and Tr1048 was treated with 10 ml each at 7-day intervals per week for 6 times. After 45 days of treatment Growth was investigated.

Strain division Average Indices(%) Tr1047 Plant height 26.8 126.4 Rootstock 31.7 142.2 Fresh weight 10.4 170.5 Tr1048 Plant height 29.4 131.8 Rootstock 33.7 151.1 Fresh weight 11.4 186.9 No treatment Plant height 21.2 - Rootstock 22.3 - Fresh weight 6.1 -

tomato( Seokwang variety )

Strain division Average Indices(%) Tr1047 Plant height 16.8 128.1 Rootstock 40.5 161.8 Fresh weight 6.42 151.2 Tr1048 Plant height 19.1 134.5 Rootstock 43.2 165.8 Fresh weight 6.54 151.2 No treatment Plant height 12.3 - Rootstock 26.0 - Fresh weight 4.5 -

pepper( Mani Ta )

Since the sclerotinia and black rot mycotic disease control agents of the present invention exhibit excellent control activity, they can be used as an effective sclerotinia and black rot fungicide prevention agent instead of chemical pesticides that have been used in the past. The sclerotinia and black rot mycotic disease controlling agent of the present invention can be used as an environmentally friendly antiseptic agent for solving soil pollution and human toxicity problems and can be used for sclerotinia and black rot mycorrhiza which have resistance against chemical pesticides.

National Academy of Agricultural Sciences KACC93157P 20120927 National Academy of Agricultural Sciences KACC93158P 20120927

Claims (5)

Sclerotium cepivorum , Botrytis cinerea , Sclerotinia sclerotiorum , Sclerotinia minor , Sclerotinia spp ., Sclerotium spp . , The growth of sclerotia and mycelium is inhibited by parasitization of sclerotia of plant pathogenic bacteria Which is characterized by having the ability to generate cellulase while having a dragging power against scleroderma and black rot mycosis,
TRICO DELMARBINES TR1047 entrusted with accession number KACC93157P or TRICO DELMASHANUM TR1048 entrusted with accession number KACC93158P.
A TRICODELMAVIRUS TR1047 strain deposited with the accession number KACC93157P of claim 1 or a TRICODELMAHANUM TR1048 strain deposited with the accession number KACC93158P and a culture solution of these strains as an active ingredient for scleroderma and black decaying scleroderma Microbial agent
The microorganism preparation according to claim 2, wherein the microbial agent further comprises at least one additive selected from the group consisting of a biopolymer, a protective agent and a nutrient.
In the CMD medium consisting of cornmeal agar and 2% dextrose, the culture temperature was 25 to 30, the pH was 5.5 to 6.5, Incubation time is from 36 hours to 52 hours,
Characterized in that the culture medium contains barley as a carbon source of the culture medium and is cultured under a cancer treatment condition.
Methods for mass production of microbial agents for sclerotinia and black rot fungi.
A method for controlling scleroderma and black decaying scleroderma, including spraying the microorganism preparation of claim 2 to plants

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