KR100442423B1 - A culture method of Shimizuomyces sp. SJM and a composition for promoting plant growth including culture solution thereof - Google Patents

Abstract

The present invention relates to a plant growth promoting composition containing Shimizuomyces sp. SJM, a culture method thereof, and a culture medium, wherein the present invention is isolated and identified as a blue-green spermatozoon fungus Mycobacterium tuberculosis ( Shimizuomyces sp. SJM, KCCM). -1024) multi-step culture in a liquid medium to cultivate the blue-green fruit worm fungus, and spray the liquid culture solution on plants such as tomatoes, peppers, tobacco, etc. There is an excellent effect of providing a composition for promotion.

Description

Cultivation method of blue-green berry fungus fungal bacteria and composition for promoting plant growth containing the culture medium {A culture method of Shimizuomyces sp. SJM and a composition for promoting plant growth including culture solution about}

The present invention relates to a plant growth promoting composition containing Shimizuomyces sp . SJM, a culture method thereof, and a culture solution. More specifically, it relates to a plant growth promoting composition that isolates and identifies the blue-green fruit sperm fungi ( Shimzu ), multistage liquid culture, and increases the growth and fruit sugar of crops containing the culture.

Indole acetic acid, gibberellin, benzylaminopurine, indolebutyric acid, etc., which are conventionally used as plant growth agents, are not only expensive as imports but also do not dissolve well in water other than gibberellin when preparing solutions. There were various problems such as to generate heat to wither plants. Therefore, there is an urgent need to develop a plant growth promoting agent that is effective in plant growth and is completely harmless to the human body.

On the other hand, Cordyceps sinensis generally refers to mushrooms that invade insects to kill insects, and then form a locus from the host. Cordyceps belongs to the taxonomy and belongs to the genus Clavicipitales (Clevicipitales). Cordyceps sinensis is a name given to the insects that enter the body of the insects in winter, absorb the nutrients, and kill the insects. However, Cordyceps sinensis has been found among insects not based on insects but based on the fruits of bacteria or plants (primary cordyceps, 淸 水 大典 성, Seongmundang Shingwangsa 381 p; Korean cordyceps, Sungjaemo 著, Kyohaksa 299 p) . On Cordyceps Formed in the Fruits of Higher Plants Kobayasi of Japan discovered Shimizuomyces paradoxa, which is based on seeds of Smilax sieboldii , and reported on their morphological characteristics. There is a bar. The inventor of the present invention, Shimizuomyces paradoxa ( Shimizuomyces paradoxa) has been published in Korean cordyceps (Seongjae Mo 著, Kyohaksa), called in Korean.

However, as described above, although the morphological characteristics of Shimizuomyces paradoxa Kobayasi have been reported, their culture and physiological characteristics have not been reported.

Therefore, the present inventors, based on the above-mentioned point, isolates the blue-green fruit nymphaechocho parasitic on the seeds of the blue-growing vine growing in Korea, and this is Shimizuomyces sp. Was deposited with the Korean spawn association on October 12, 2001 and was given accession number KCCM-10324. The culture and physiological characteristics of the strain were investigated, and then the culture of the strain was applied to the fruits and crops, and the culture was sprayed on the fruits and vegetables to increase the sugar content of the vegetables, and confirmed that the growth of the crop was completed. .

The inventors of the present application No. 00-47398 (liquid seed culture method and apparatus for mass production of Cordyceps fruit fruit body) inoculation device 10 and liquid seedling device 20 for aseptic inoculation and liquid culture. It was initiated. In the present invention, the apparatus was used for the sterile inoculation and liquid culture of the Shimizuomyces sp. SJM, as shown in FIGS. 1 and 2.

An object of the present invention is to provide a blue-green fruit sperm fungi ( shimizuomyces sp. SJM).

And another object of the present invention to provide a method for culturing the blue-green fruit spermatozoa fungus.

And it is still another object of the present invention to provide a composition for promoting plant growth, which contains a culture solution of the blue-green berry fungus subtilis, and raises the sugar content in the growth and fruit of crops.

The object of the present invention is to deposit the blue-green fruit sperm fungi supernatant isolated from Obong-san, Gangwon-do, Korea, to investigate the physiological and culture characteristics of the strain, and to expand and culture the strain in a solid medium, the expanded culture Using the inoculation device 10 and the liquid culture device 20 shown in Figs. 1 and 2 to produce a liquid culture material by multi-step cultivation of 100 ml, 2 L 8 L, and then the liquid culture material was cropped. This was achieved by confirming the increase in crop growth and sugar content by spraying on.

Hereinafter, the configuration of the present invention will be described.

Figure 1 shows a preferred inoculation device 10 for aseptically performing the liquid seed inoculation of the blue-green fruit caterpillar fungus of the present invention.

Figure 2 shows a preferred liquid seed culture apparatus 20 for facilitating the inoculation, incubation and discharge of the liquid seed of the present invention.

* Description of the symbols for the main parts of the drawings *

10) Inoculation device 11) Inoculation container

12) Plug 13) Air injection pipe

14) Air Filter 15) Discharge Pipe

20) Liquid spawn incubator 21) Culture vessel

22) Plug 23) Combined pipe

24) Locking device 25) Air filter

26) Air exhaust pipe 27) Filter

Figure 3 shows the manufacturing process of the plant growth promoting composition containing a liquid culture solution of the present invention blue-green fruit chung fungi.

Figure 4 is a photograph after 10 days of cultivation of tobacco (a) and sprayed tobacco (b) not sprayed with the plant growth promoting composition containing the present invention blue-green fruit berry fungus culture.

a) unsprayed cigarettes b) sprayed cigarettes

Figure 5 is a photograph after 45 days of cultivation of tobacco (a) and sprayed tobacco (b) that did not spray the plant growth promoting composition containing the present invention blue-green berry fungus Chungchosa culture.

a) unsprayed cigarettes b) sprayed cigarettes

Figure 6 is a photograph after 40 days of cultivated Chinese cabbage (a) and sprayed Chinese cabbage (b) that did not spray the plant growth promoting composition containing the present invention blue-green berry fungus Chungchocho.

a) unsprayed cabbage b) sprayed cabbage

Figure 7 is a photograph of the ground portion of the rice after cultivation of rice (a) and sprayed rice (b) that did not spray the plant growth promoting composition containing the present invention blue water berry fungus Chungchocho culture in the mother bed.

a) ground portion of unsprayed rice b) ground portion of sprayed rice

Figure 8 is a photograph of the basement of the rice after cultivation of rice (a) and sprayed rice (b) that did not spray the plant growth promoting composition containing the present invention blue-green berry fungus Chungchocho culture in the mother bed.

a) underground of unsprayed rice b) underground of sprayed rice

The present invention comprises the steps of separating and identifying strains by separating the Cordyceps sinensis collected from Obong-san, Gangwon-do to investigate the morphological, culture, and physiological characteristics of the strains; Inoculating the strain in a solid medium consisting of SDAY (dextrose, yeast extract, peptone, agar and distilled water) and incubating for 20 to 30 days in a thermostat at 22 to 30 ° C; For inoculum culture, 100 ml of inoculum medium consisting of water, dextrose, yeast extract, malt extract, and peptone was prepared and placed in the inoculation vessel 11 of the inoculation device 10 shown in FIG. Inoculating one blue-green berry fungus Choong-chung and incubating at 22-30 ° C. for 20-30 days; Preparing a 2 L liquid medium consisting of water, dextrose, malt extract, yeast extract, peptone, and an antifoaming agent for liquid seed culture of the present invention; The 2 liter liquid medium prepared in the above step was placed in the culture vessel 21 of the liquid culture apparatus 20 shown in FIG. 2, and 100 ml of the inoculum culture solution was discharged into the liquid seed culture apparatus 20 at 0.3 to 30 ° C. incubating for 20 to 30 days while injecting sterile air of about vvm; By discharging the culture solution of the above step 400ml using a compressor, inoculated into 8 liter liquid medium (water, brown sugar, lactorox, antifoaming agent) prepared for mass cultivation of blue sugar spermatozoon fungus bacteria and 0.3vvm at 22 ~ 30 ℃ Culturing for 20-30 days while injecting sterile air to an extent; And irrigation and foliar spraying before planting at a ratio of 1.500 ml of water: 1 ml of blue-green spermatozoon fungus supernatant culture cultured as described above, and foliar spraying is performed again a week later. After 15 days, spray again on the foliar surface.

The present invention is to isolate and identify shimizuomyces sp. SIM based on the seeds of Smilax sieboldii in Obongsan, Gangwon-do, and to investigate their physiological and cultural characteristics, 2001 Deposited October 12, 1989, accession number KCCM-10324 was assigned.

Inoculation of the blue-green berry fungus Choong-choong for the inoculum cultivation in the present invention, when the mycelium grows in about 70% in Petri dishes, inoculates the mycelium pieces with platinum ball so that other bacteria do not enter the culture medium in the culture vessel. The larger the number, the shorter the incubation period of the inoculum, and the higher the density of the inoculated mycelium, the smaller the diameter of the mycelium mass after culture. The inoculum culture was incubated for 20 to 30 days at 22 to 30 ° C, preferably at 25 ° C for 25 days.

In the same blue-green fruit caterpillars, the incubation period tended to be shorter as the culture temperature approached the optimum temperature condition of the mycelia. When the inoculum was inoculated into the liquid culture solution of the above step, the liquid culture solution: the culture inoculation source was 20 to 200: 1, and preferably 20: 1. In addition, the culture of the liquid spawn of the step was incubated by injecting the sterilized air at 0.1 to 1.0vvm for 0 days at 22 to 30 ℃, preferably 20 to 30 while injecting the sterilized air of about 0.3vvm at 25 ℃ Incubated daily.

The 1000 ml solid medium used for the expansion of blue-green berry fungus fungi was composed of 10 to 80 g of dextrose, 2 to 20 g of yeast extract, 2 to 20 g of peptone, 10 to 20 g of agar and distilled water, preferably 20 g of dextrose, It consists of 5g of yeast extract, 5g of peptone, 20g of agar and distilled water.

The liquid culture solution of the blue-green berry fungus Chungchocho is prepared through a multi-step culture. That is, the first step is to incubate the culture of the blue-green fruit worm fungus supernatant grown in solid medium in 100ml of inoculum medium. The inoculum medium is composed of dextrose 0.5-4g, yeast extract 0.1-1g, malt extract 0.1-1g, peptone 0.1-1g and distilled water, preferably dextrose 1g, yeast extract 0.3g, malt extract 0.3g, It consists of 0.5 g of peptone.

The second step consists of 10 to 80 g of dextrose, 2 to 20 g of yeast extract, 2 to 20 g of malt extract, 2 to 20 g of peptone, 2 to 10 ml of antifoam, and distilled water, preferably 20 g of dextrose, 6 g of yeast extract, and malt extract. The culture is inoculated by inoculating the culture medium into a 2 L liquid medium consisting of 6 g, 10 g of peptone, 3 ml of antifoam, and distilled water. Defoamers added to prevent foaming when air is introduced are preferably cooking oil, cottonseed oil, castor oil, corn oil, olive oil and palm oil, and corn oil is most preferred.

The third step is to prepare an 8 L liquid medium consisting of 750-2250 g of brown sugar, 50-150 g of lactohyrox, 3-15 ml of antifoam, and distilled water, preferably 1500 g of brown sugar, 100 g of lactohyrox, 5 ml of antifoam, and distilled water. Inoculate the culture culture in the step is to incubate in large quantities.

Fruit vegetables, preferably tomato, pepper, irrigation and foliar surface of the present invention blue berry caterpillar vinegar culture solution 1ml: water sprayed before planting at a rate of 1.5 ℓ, foliar spraying a week later, and foliar surface after 15 days Again spraying increased sugar content. In addition, spraying crops, preferably tobacco, cabbage or rice, in the same manner as described above, promoted growth, robustness and increased yield. The spreading amount or the spreading method may be modified within the range commonly used in the art.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Preferred inoculation apparatus and liquid seed culture apparatus used to aseptically inoculate and culture the blue-green berry fungus supernatant of the present invention are as shown in FIGS. 1 and 2. In the present invention, the liquid culture is a multi-step culture, so the liquid seed culture apparatus is not shown in FIG. 2, but the 2 L container and the 8 L container are continuously connected.

Example 1 Isolation and Identification of Blue Fruit Berry Insects

The fruiting bodies of Cordyceps sinensis, collected at Obong-san, Gangwon-do, were fixed with a tape of a petri-dish cap containing water agar, and then covered with a lid. Aspergillus spores germinated on water agar medium were transferred to potato agar medium (PDA) under a microscope, which was called SJM.

Experimental Example 1 Investigation of Morphological Characteristics of Isolated Bacteria

The isolated SJM is parasitic to the seeds of Smilax sieboldii and forms 1 to 4 loci, the surface of the seed is covered with white mycelia, the locus is grayish gray, and the pericardium is buried. It is densely distributed. It consists of a 27mm × 2-5mm head and a 52mm × 10mm bag. Western pear-shaped sulcus angle is 350-370㎛ × 160-200㎛, with 4-8 porcine vesicles combined, and vesicles are 60 ~ 70㎛ long spindle, bulging central cell, outer wall It consists of an inner wall. Asystole spores do not germinate as secondary spores.

Experimental Example 2 Investigation of Culture Characteristics of Isolated Bacteria

The isolated SJM strains were inoculated in SDAY (dextrose, yeast extract, peptone, agar and distilled water) medium, and then cultured in a 24 ± 1 ° C. thermostat and used as inoculum.

1) Selection of basic medium suitable for mycelial growth

In order to select a basic medium suitable for mycelial growth of SJM strains, a synthetic medium having a medium composition as shown in Table 1 below was autoclaved at 121 ° C. for 1.2 minutes at 1.2 psi for 20 minutes, and then dispensed in 15 to 20 ml in sterile Petri dishes (87ø × 15 mm). The mycelial tip of the inoculated SJM strain cultured in the above was cut out with a 5 mm diameter cork borer, moved to the center of the petri dish, and grown in a thermostat at 24 ± 1 ° C for about 55 days. And the color was investigated and the result is shown in Table 2.

Composition of Medium for Investigating Culture Characteristics of SJM Strains Medium (g / ℓ) PDA SDAY MYA YMA MCM MM MPDA HM CDA PSA BM potato 200 200 Dextrose 20 20 10 20 10 20 Sucrose 30 20 30 Malt Extract 20 3 20 Yeast extract 5 2 3 2 3 3 Peptone 5 5 5 MgSO ₄ 0.5 0.5 0.5 One KH ₂ PO ₄ 0.46 One One K ₂ HPO ₄ One One Ebiose 5 Hyponex 3 KCl 0.5 NaNO ₃ 3 FeSO ₄ 0.01 Agar 20 20 20 20 20 20 20 20 20 20 20

Growth and density of mycelia according to medium of SJM strains, and color badge SDAY YMA MYA MM MCM HM CDA MPDA PDA PSA BM Colony diameter (mm / 55days) 26.8 25.5 36.0 31.4 38.3 27.2 32.5 34.5 29.4 23.2 26.0 Density ^* +++ ++ + + +++ ++ +++ ++ +++ +++ +++ Color ^** Y.B B. P.Y Y.W G.Y O.B W. Y.W Y.G Y.G P.Y * +: Lean ++: normal +++: dense ** YB: yellowish brown, B: brown, PY: pale yellow, YW: yellowish white , GY: greyish yellow, OB: olive brown, W: white, YG: yellowish grey

As shown in Table 2, SJM strains showed the best mycelial growth in the order of MCM, MYA, MPDA medium, MCM medium was also most suitable in terms of density.

(2) optimum temperature survey

The MCM medium selected above was autoclaved at 121 ° C. at 1.2 psi for 20 minutes, and then dispensed in a sterilized Petri dish (87 ° × 15 mm) by 15-20 ml each to view the mycelial tips of the inoculated SJM strains. After cutting with a cork borer, the cells were placed in the center of the petri dish and cultured for 55 days in a thermostat at 15, 20, 25, 30, and 35 ° C. As shown in Table 3 below, the optimum temperature was found to be 25 ° C.

Growth and density of mycelia depending on temperature of SJM strains, and color Temperature (℃) 15 ℃ 20 ℃ 25 ℃ 30 ℃ 35 ℃ Colony diameter (mm / 55days) 35.5 44.5 55.5 21.3 0 Density ^* +++ +++ +++ +++ × Color ^** Y.G Y.G O.G Y.G × * +: Lean, ++: normal, +++: dense. ** Y.G: yellowish grey, O.G: orange grey

Experimental Example 3 Investigation of Physiological Characteristics of Isolated Bacteria

In order to select suitable nutrient sources for mycelial growth, MPDA, a synthetic medium, is used as the basic medium, and carbon sources include four monosaccharides such as dextrose, three disaccharides such as sucrose, and two polysaccharides such as dextrin. A total of 9 carbon source concentrations were prepared so as to be the same carbon source as 10 g of dextrose, and autoclaved at 121 ° C. (1.2 psi) for 20 minutes, and then inoculated with 5 mm diameter mycelium and incubated in a thermostat at 25 ° C. for 55 days. The growth and density and color of mycelia were examined and the results are shown in Table 4 below.

Nitrogen sources consist of six inorganic nitrogen sources, such as ammonium tartrate, three organic nitrogen sources, such as peptone, and three amino acids, such as glycine, with a total concentration of 12 nitrogen sources equal to 5 g of peptone. To adjust the nitrogen content to prepare a medium and cultured in the same manner as above to examine the growth rate and density and color of the mycelia and the results are shown in Table 5 below.

Inorganic salts were prepared by adding 0.05% (w / v) of 12 inorganic salts other than CaCO ₃ to prepare a medium and incubating the same method as described above to investigate the growth, density and color of mycelia. Shown in

In addition, to test the ratio of carbon and nitrogen sources suitable for mycelial growth, dextrose is used as the carbon source and peptone is used as the nitrogen source. 40: 1, 20: 1, 10: 1, 5: 1, 2: The medium was prepared by adjusting to 1, 1: 1, 1: 1.5, and cultured in the same manner as above to examine the growth degree and density and color of the mycelia, and the results are shown in Table 7 below.

Growth and density of mycelia depending on the type of carbon source of SJM strains, and color Carbon sources ^* N C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 Colony diameter (mm / 55days) 35.3 52.0 45.7 11.3 42.8 50.0 42.5 42.2 48.0 38.2 Density ^** + +++ +++ + +++ +++ ++ +++ + + Color ^*** B.O Y.W Y.G L.Y Y.G P.Y G.Y G.Y B.O O.G * N: no addition, C-1: dextrose, C-2: fructose, C-3: Xylose, C-4: Mannose, C-5: Sucrose, C-6: Lactose, C-7: Maltose, C-8: Dextrin, C-9: Starch ** +: Lean, ++: Normal, ++ +: Dense *** BO: brownish orange, YW: yellowish white, YG: yellowish grey, LY: light yellow, PY: Pale yellow, GY: greyish yellow, OG: orange grey

Growth and density of mycelia depending on the nitrogen source of SJM strains, and color Nitrogen sources ^* N N-1 N-2 N-3 N-4 N-5 N-6 N-7 N-8 N-9 N-10 N-11 N-12 Colony diameter (mm / 55days) 45.5 54.7 45.5 53.5 36.7 45.5 34.0 49.5 55.0 39.8 26.3 34.7 39.5 Density ^** + ++ ++ ++ ++ ++ ++ ++ +++ ++ ++ ++ ++ Color ^*** W Y.W G.Y Y.G G.O Y.W G.Y Y.G Y.G Y.G G.Y Y.G G.Y * N: no additive, N-1: peptone, N-2: yeast, N-3: ammonium tartrate, N-4: ammonium nitrate, N-5: Ammonium phosphate, N-6: Ammonium sulfate, N-7: Sodium nitrate, N-8: Potassium nitrate, N-9: Tryptophan (Typtone), N-10: Glycine, N-11: Asparagine, N-12: Alanine ** +: Lean, ++: Normal, +++: Dense ** * W: white, YW: yellowish white, GY: greyish yellow, YG: yellowish grey, GO: greyish orange

Growth and density of mycelia according to the inorganic salts of SJM strains, and color Mineral salts ^* N M-1 M-2 M-3 M-4 M-5 M-6 M-7 M-8 M-9 M-10 M-11 M-12 M-13 Colony diameter (mm / 55days) 4.72 5.08 4.82 4.20 3.87 5.83 5.73 5.00 3.93 4.80 5.23 4.77 5.03 3.38 Density ^** + ++ ++ ++ + + +++ ++ ++ ++ ++ + + + Color ^*** Y.G Y.G O.B Y.G Y.B Y.G Y.G G.Y O.B Y.G Y.G Y.G Y.G O.B N: untreated, M-1: CaCO ₃ , M-2: CaCl ₂ , M-3: CaSO ₄ 1 / 2H ₂ O, M-4: FeSO ₄ 7H ₂ O, M-5: KH _{2 PO 4, M-6: K} 2 HPO 4, M-7: MgSO 4 · 7H2O, M-8: ZnSO 4 · 7H 2 O, M-9: NaSO 4 M-10: MnSO 4, M-11: NaCl , M-12: KCl, M-13: CuSO ₄ 5H ₂ O. ** +: lean, ++: normal, +++: dense. *** YG: yellowish grey, OB : Olive brown, YB: yellowish brown, GY: greyish yellow

Growth and density of mycelia according to C / N ratio of SJM strains, and color C / N ratio 40: 1 20: 1 10: 1 5: 1 2: 1 1: 1 1: 1.5 Colony diameter (mm / 55days) 3.30 3.72 4.55 5.13 6.00 5.28 4.42 Density ^* ++ +++ +++ +++ ++ + ++ Color ^** Y.G Y.G G.Y P.Y Y.G Y.G Y.G * +: Lean, ++: normal, +++: dense ** Y.G: yellowish grey, G.Y: greyish yellow, P.Y: pale yellow

As shown in Table 4, the relationship between the mycelial growth and the density was found to be a suitable carbon source in the order of dextrose and sucrose. In addition, as shown in Table 5, the suitable nitrogen source was found in the order of potassium nitrate, peptone. And, as shown in Table 6, when looking at the relationship between the mycelial growth and density, K ₂ HPO ₄ was found to be a suitable inorganic salts. As shown in Table 7 above, C / N showed the best growth at 5: 1 when considering the relationship between mycelial growth and density.

Experimental Example 4 Identification of Isolated SJM

SJM has the same morphological characteristics as in Experimental Example 1, and is based on seeds of Smilax sieboldii , and was identified as Shimizuomyces sp. Deposited on the 12th of February, he was assigned accession number KCCM-10324.

Example 2: Cultivation of Blue Fruit Berry Cordyceps

SDAY (20 g of dextrose, 5 g of yeast extract, 5 g of peptone, 20 g of agar, distilled water) was inoculated with blue-green fruit caterpillar ( Shimizuomyces sp.SJM, KCCM-10324) in a solid medium and expanded for 25 days in a thermostat. Incubated. Extended cultured blue-green fruit nymphaechocho ( Shimizuomyces sp.SJM, KCCM-10324) was cultivated in bulk through a three-step liquid culture process as follows.

First step: cultivation of blue berry fruit Chungcho Inoculation source

In order to incubate the inoculum of the blue-green fruit spermatozoa fungus ( Simizuomyces sp.SJM, KCCM-10324), a culture solution was prepared as shown in Table 8 below and sterilized at 121 ° C. for 20 minutes using an autoclave.

In order to prevent contamination of various bacteria, the inoculation device 10 as shown in Figure 1 was used. The inoculation mechanism 10 is composed of an inoculation vessel 11, a stopper portion 12, an air inlet tube 13, an air filter 14, and a culture medium discharge tube 15. The stopper portion 12 of the inoculation container 11 is connected to the air inlet pipe 13 and the culture medium discharge pipe (15). The air injection pipe 13 is equipped with an air sterilizing air filter 14, one end of which is located at the bottom of the inoculation vessel 11, the other end is connected to the compressor. One end of the culture liquid discharge pipe 15 is located at the bottom of the inoculation vessel 11, and the other end thereof is connected to the combined pipe 23 installed in the liquid seed culture device 20. The culture medium discharge pipe 15 was connected to each other by varying the size of the tube, and the rubber pipes were connected and then covered with a foil to prevent contamination of various bacteria. Installed in 1cm.

100 ml of the inoculum medium shown in Table 8 below was prepared and placed in the inoculation vessel (11) and inoculated with the expanded cultured blue-green fruit worm fungus supernatant and incubated at 25 ° C for 25 days.

Composition ratio of medium for culturing liquid seedlings ingredient 100 ml (inoculation medium) 2ℓ liquid medium 8ℓ liquid medium Dextrose 1 g 20 g Malt Extract 0.3 g 6 g Filial seed extract 0.3 g 6 g peptone 0.5g 10 g Brown sugar 1.5 kg Lactohilux 100 Corn oil 3 ml 5 ml

Second step: liquid seed culture step

Cultivation of the liquid seed was used for the liquid seed culture apparatus 20 as shown in FIG. The liquid seed culture device 20 is a culture vessel 21, the stopper 22, the combined pipe 23, the locking device 24, the air filter 25, the air discharge pipe 26 and the filter 27 Consists of. The culture vessel 21 accommodates a liquid culture solution, and the stopper 22 at the top of the culture vessel has a combined pipe 23 and an air discharge pipe 26 which serve to discharge the culture solution after the air, the inoculation source injection and the culture are completed, respectively. ) Is connected. The combined pipe 23 is equipped with a lock 24 to control the sealing of the tube and the air filter 25 for air disinfection, and the air discharge pipe 26 is discharged to the outside of the detoxifying harmful bacteria that may be generated in the incubator The filter 27 which prevents it is mounted. One end of the combined pipe 23 is located at the bottom of the culture vessel 21 and the other end is connected to the compressor to supply air into the culture vessel 21. One end of the air discharge pipe 26 is connected to the inside of the culture vessel 21, and the other end is open to the outside atmosphere. Although not shown in FIG. 2, the culture device 20 is connected to the culture device by varying the size of the culture vessel.

2 L of the liquid medium of Table 8 is prepared and placed in the culture vessel 21. The capacity of the culture vessel 21 in the second stage is 2.5 liters. When inoculating the inoculum culture medium cultured in step 1 into the liquid seed culture device 20, the air is injected into the air injection pipe 13 using a compressor, and the culture medium discharge pipe 15 is connected to the liquid seed culture device 20. The inoculum culture liquid was discharged to the liquid seed culture device 20 by the pressure of the air introduced into the air inlet pipe 13 by connecting to the combined pipe 23. The temperature was maintained at 25 ℃, and cultured for 25 days while injecting the sterilized air of about 0.3 vvm to obtain a liquid seed.

Step 3: Mass Culture of Liquid Seedling

Although not shown in FIG. 2, as described above, a culture device having a different capacity (10 L) of the culture vessel is connected to the culture device 20 of the second stage. That is, the 8 L liquid medium shown in Table 8 is prepared and placed in the 10 L culture vessel. Then, the liquid spawn cultured in step 2 was discharged, and 400 ml of the culture solution was inoculated into the 8 L liquid medium in the same manner as in the step 2 above. And the temperature was maintained at 25 ℃ and cultured for 25 days while injecting sterilized air at 0.3vvm speed to prepare a liquid culture solution.

Example 3: Effect of the liquid culture material of the blue-green fruit caterpillar fungus on the growth and sugar content of plants

The growth and sugar content of the liquid culture solution of Shimizuomyces sp.SJM, KCCM-10324 prepared in Example 2 were sprayed on foliar and irrigation on tomatoes, peppers, tobacco, cabbage or rice. Observed.

Experimental Example 5: Determination of the sugar content of tomato sprayed with blue-green fruit berry fungus culture solution of the present invention

First, before planting blue liquid lychee cultivated vinegar ( Shimizuomyces sp. SJM, KCCM-10324) liquid culture medium in a multi-stage liquid culture of Example 2 in a ratio of 1.5 L of water: 1 ml of blue berry edible fungus culture medium Once a week, sprinkle three times a day after 15 days, and then cultivated for about 40 days to measure the sugar content, and the results are shown in Table 9 below. As shown in Table 9 below, the average value of 6.41 and 5.40 non-treated groups was 6.41, and the sugar content of the treated group was about 1 higher. In addition, the treated group was observed to be more robust than the untreated tomato.

Sugar content measurement results of tomato and untreated tomato treated with blue-green fruit berry fungus culture solution of the present invention One 2 3 4 5 6 7 8 9 10 Average Treatment 5.5 5.7 6.0 6.7 6.0 6.8 7.0 6.9 6.6 6.9 6.41 No treatment 4.7 4.5 5.0 5.0 5.4 5.6 6.2 6.3 5.6 5.7 5.40

Experimental Example 6: Investigation of the quantity and weight of red pepper sprayed with the present invention blue fruit berry fungus

Blue-green fruit berry fungus ( Simizuomyces sp. SJM, KCCM-10324) cultured in a multi-stage liquid culture of Example 2 was sprayed in the same manner as in Experimental Example 5 to the pepper and cultivated for about 40 days and the weight of the pepper The number per group was measured and the results are shown in Table 10 below. As shown in Table 10 below, the average number of treatments of the blue-green fruit Cordyceps sinensis culture medium was 37.4, the number of untreated was 32.8, and the number of treatments was about five. In weight, 417g was treated as a treatment, and 349g was treated as an untreated, showing a difference of about 70g. In addition, as in tomato, the treatment group was observed to grow stronger for longer periods than the pepper in the untreated group.

Weight and quantity measurement results of red peppers and untreated red peppers treated with the blue-green berry fungus Cultivation medium of the present invention One 2 3 4 5 total Average Treatment Quantity 40 40 34 40 33 187 37.4 weight 390 410 415 450 420 2085 417 No treatment Quantity 34 32 29 40 29 164 32.8 weight 335 350 340 380 340 1745 349

Experimental Example 7 Investigation of the Length and Width of Tobacco Leaves Sprayed with Blue-Crown Fruit Insecticide

Shimizuomyces sp. SJM, KCCM-10324 cultured in a multi-step liquid culture of Example 2 sprayed on the tobacco in the same manner as in Experiment 5, and then grown for about 45 days tobacco leaf length And the width is measured and the results are shown in Table 11 below. As shown in Table 11 below. Tobacco leaves were 75cm in average, 60cm in untreated group, 15cm long in leaves, and 55cm in average and 45cm in average, and 10cm in width. In addition, as shown in Fig. 4 (cultivated about 10 days) and Fig. 5 (cultivated about 45 days), in the untreated group (a) there were many trees showing viral symptoms and growth was not good, but the treated group (b) showed virus symptoms. There was no trees, and it grew well.

Measurement results of the length and width of the leaves of tobacco and untreated tobacco treated with blue-green fruit berry fungus culture of the present invention One 2 3 4 5 6 7 8 9 10 Average Treatment 80 × 60 74 × 55 65 × 50 75 × 60 72 × 54 82 × 56 77 × 50 79 × 59 73 × 52 73 × 54 75 × 55 No treatment 64 × 48 59 × 42 60 × 45 70 × 55 55 × 40 58 × 44 55 × 42 61 × 46 60 × 47 58 × 41 60 × 45

Experimental Example 8: Investigation of the growth of Chinese cabbage sprayed with the present invention blue fruit berry fungus

Growth of Chinese cabbage after cultivation for about 40 days by spraying the liquid culture solution of blue-green fruit berry fungus ( Simizuomyces sp.SJM, KCCM-10324) cultured in the multi-step liquid culture of Example 2 in the same manner as in Experiment 5 The degree was observed and the result is shown in FIG. As shown in Figure 6, it was observed that the growth of the blue-green berry caterpillar culture medium according to the present invention (b) is vigorous growth compared to the case of (a) not spraying.

Experimental Example 9: Investigation of the growth of rice sprayed with the present invention blue fruit berry fungus

Shimizuomyces sp. SJM, KCCM-10324 liquid culture medium cultured in the multi-stage liquid culture of Example 2 was sprayed on rice in the same manner as in Experiment 5 to grow the length of rice grown for about 20 days The growth rate was measured and the results are shown in Table 12 below. As shown in Table 12, the average number of the treated chunggae sperm Cordyceps sinensis culture medium was 12.46cm, the untreated group was 8.16cm to grow about 4cm more. In addition, the growth degree of the ground and underground parts of the rice is shown in Figs. As shown in Figures 7 and 8, the growth of the ground and the underground (root) was more vigorous compared to the case of spraying the blue-green berry worm fungus culture medium (b) was not sprayed (a).

Growth Length Measurement Results of Rice and Untreated Rice One 2 3 4 5 Average Treatment 12 13.5 13 11 12.8 12.46 No treatment 8.5 8 9 7.5 7.8 8.16

As described above, the present invention, as described through Examples and Experimental Example, isolates and identifies the blue-green fruit spermatozoa fungi ( Shimzuomyces sp.SJM, KCCM-10324) based on seeds of the blue-green vines, and liquid culture medium It is effective in mass cultivation of Shimizuomyces sp. SJM, KCCM-10324 by multi-stage cultivation at, and the sugar content and growth of the plant are increased when the liquid culture solution is sprayed on plants such as fruits, fruits, and crops. Since it is effective, it provides an environment-friendly composition for promoting plant growth without toxicity, which is a very useful invention for the biopesticide industry.

Claims (4)

delete Shimizuomyces sp. SJM (KCCM-10324) was prepared in 10 to 80 g of dextrose, 2 to 20 g of yeast extract, 2 to 20 g of peptone, 10 to 20 g of agar, and 22 to 30 ° C. in 1 L solid medium. Expanding culture for 20-30 days; Inoculated in the blue-green berry fungus fungi expanded in this step in 100ml medium consisting of dextrose 0.5 ~ 4g, yeast extract 0.1 ~ 1g, malt extract 0.1 ~ 1g, peptone 0.1 ~ 1g and distilled water 20 at 22 ~ 30 ℃ Incubating for 30 days; The anti-foaming agent selected from the culture medium cultured in the above step 10 ~ 80g, yeast extract 2 ~ 20g, malt extract 2 ~ 20g, peptone 2 ~ 20g, domestic cooking oil, cottonseed oil, castor oil, corn oil, olive oil and palm oil Inoculating 2 l medium consisting of 2-10 ml and distilled water and incubating at 22-30 ° C. for 20-30 days; And The culture cultured in the above step is inoculated into 8 liter medium consisting of brown sugar 750-2250 g, lactohyrox 50-150 g, household cooking oil, cottonseed oil, castor oil, corn oil, olive oil and palm oil, any one antifoaming agent 3-15 ml and water. Method of culturing blue-green berry fungus fungi comprising the step of culturing at 20 to 30 days at 22 to 30 ℃. Plant growth promoting composition for increasing the growth and fruit sweetness of crops, characterized in that it contains a culture solution of Shimizuomyces sp. SJM, KCCM-10324. According to claim 3, The plant is a plant growth promoting composition, characterized in that any one selected from the group consisting of tomatoes, peppers, tobacco, cabbage, rice.