KR101167865B1 - Medium composition for pure culture of Perilla rust fungi - Google Patents

Abstract

The present invention is ammonium phosphate (NH ₄ H ₂ PO ₄ ), boric acid (H ₃ BO ₃ ), calcium chloride (CaCl ₂ ), copper sulfate pentahydrate (CuSO ₄ ~ 5H ₂ O), ethylenediaminetetraacetic acid disodium dihydrate (Na ₂ EDTA? 2H ₂ O), iron sulfate heptahydrate _{(FeSO 4? 7H 2 O)} , magnesium sulfate (MgSO _4), manganese sulfate monohydrate _{(MnSO 4? H 2 O)} , potassium iodide (KI), potassium nitrate ( KNO ₃ ), zinc sulfate heptahydrate (ZnSO ₄ ˜7H ₂ O), molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ ˜2H ₂ O), Coleosporium plectranthi , including agar and sucrose The present invention relates to a method for purely cultivating sesame leaf rust fungus such as cholose porium plectranti using the artificial medium composition for pure culture of sesame leaf rust fungus, such as the artificial medium composition, the solid medium prepared using the artificial medium composition, and the solid medium.

Description

Medium composition for pure culture of Perilla rust fungi

The present invention relates to an artificial medium composition for pure culture of sesame leaf rust fungus, such as Coleosporium plectranthi , more specifically ammonium phosphate (NH ₄ H ₂ PO ₄ ), boric acid (H ₃ BO ₃ ), Calcium chloride (CaCl ₂ ), copper sulfate pentahydrate (CuSO ₄ ~ 5H ₂ O), ethylenediaminetetraacetic acid disodium dihydrate (Na ₂ EDTA? 2H ₂ O), iron sulfate heptahydrate (FeSO ₄ ? 7H ₂ O) , Magnesium sulfate (MgSO ₄ ), manganese sulfate monohydrate (MnSO ₄ ? H ₂ O), potassium iodide (KI), potassium nitrate (KNO ₃ ), zinc sulfate heptahydrate (ZnSO ₄ ? 7H ₂ O), sodium molybdenum oxide Artificial medium composition for pure culture of sesame leaf rust fungus, such as Coleusporium plectranthi , including dihydrate (Na ₂ MoO ₄ ˜2H ₂ O), agar and sucrose, manufactured using the artificial medium composition Using a solid medium and the solid medium, such as choleosporium plectranti It relates to a method of pure culture of sesame leaf rust fungus.

Rust fungus is an obligate parasite that gets nutrients from plants and multiplies in plants to complete life history. About 7,000 rust fungi are known as parasites of cereals, legumes and other plants (ferns, monocots and dicotyledons). Life history of rust fungus can be clearly divided into two to five spore stages. Some rust fungus (hetroceious) alternates with two taxonomically unrelated hosts to complete life history, while some rust fungus (autoceious) has only one host. in need.

Sesame leaf ( Perilla frutescens var . Japonica Hara.) Is an annual plant native to Asia belonging to the labiatae. In Korea, sesame leaves are grown in open fields and greenhouses. Seeds of sesame leaves are used as cooking oil and also used directly as a dry powder for seasoning. In addition, sesame leaves are mainly cultivated in open fields and greenhouses for use as vegetables and for eating meat cooked with seasoning and flavoring ingredients, ie for reproduction. In Korea, the seed production area of sesame leaves is about 24,000 ha (Anonymous, 2006, Agricultural & Forestry Statistical Yearbook 2006 , Ministry of Agriculture & Forestry, Republic of Korea. p. 319), the area for growing vegetables is estimated at about 850 ha. In sesame leaves, most leaf diseases (spots, gray mold, anthrax, rust, etc.) damage crops and are very common (Kim et al., 2001, Plant Pathol. J. 17: 236-241). Of these, rust is a very serious disease and widespread in Korea.

In open fields and greenhouses, sesame leaf rust occurs at any time during the growing season (late June to mid-August). In the early stages of rust, very small yellow bumps appear on the lower surface of the leaf, which usually spreads inward from the edge of the leaf, accompanied by yellow-brown stains formed on the opposite upper side, and within 2 to 3 weeks the spore mass is released. Cover the whole. Enlarged pustules may initially appear as yellow spore masses, but later turn white. The leaf tissues dry out and die. Infection usually begins in the lower leaves and spreads upward, making the whole plant appear dusty (due to rust spores). The rust spore mass appears as yellow to orange (uredinia and uredospore).

The inability to cultivate the rust fungus in vitro has given difficulties to the study of phytopathologists regarding rust control. Pure culture of rust fungus in the laboratory can be of great help in controlling the disease.

The inventors have succeeded for the first time in vitro cultivation of perilla leaf rust fungus such as Coleosporium plectranthi causing perilla leaf rust. The present invention enables the in vitro study of the most important stages in the ecology of sesame leaf rust fungus, such as cholesteropium plectranti, which is potentially applicable to other hemi-biotrophic or biocidal fungi. It may be.

Korean Patent No. 10-0445163 discloses a selection medium composition for pure culture of microorganisms in a semi-flow, high salt, acidic condition.

The present invention is derived from the above requirements, the present inventors have found a medium composition capable of pure culture of sesame leaf rust fungi such as Coleosporium plectranthi , which is a sesame leaf rust fungus, and the mold is well The present invention has been completed by identifying the optimum temperature for growth.

In order to solve the above problems, the present invention is ammonium phosphate (NH ₄ H ₂ PO ₄ ), boric acid (H ₃ BO ₃ ), calcium chloride (CaCl ₂ ), copper sulfate pentahydrate (CuSO ₄ ~ 5H ₂ O), ethylenediaminetetra Disodium acetate dihydrate (Na ₂ EDTA? 2H ₂ O), iron sulfate heptahydrate (FeSO ₄ ? 7H ₂ O), magnesium sulfate (MgSO ₄ ), manganese sulfate monohydrate (MnSO ₄ ? H ₂ O), potassium iodide (KI), potassium nitrate (KNO _3), zinc sulfate heptahydrate _{(ZnSO 4? 7H 2 O)} , water _{(Na 2 MoO 4? 2H 2} O) molybdenum sodium oxide dihydrate, kolreoh containing agar and sucrose sports Solarium Provided is an artificial medium composition for pure culture of sesame leaf rust fungus such as Coleosporium plectranthi .

The present invention also provides a solid medium prepared using the artificial medium composition.

In addition, the present invention provides a pure culture method of sesame leaf rust fungus, such as cholose porium plectranti comprising the step of inoculating and inoculating the sesame leaf rust fungus spores, such as cholose porium plectranti in the solid medium.

In the present invention, the artificial medium composition for pure culture of sesame leaf rust fungus such as Coleosporium plectranthi provides an opportunity to study the fungus in vitro ( in vitro ) to prevent the sesame leaf rust. It may be helpful to control.

Figure 1 shows a colony (A) and an enlarged view (B) of Coleosporium plectranthi , a type of sesame leaf rust fungus, grown in artificial solid culture medium.
FIG. 2 shows the spores (left view) collected from the agar plate and viewed under an optical microscope, and the spores and hypospores (right view) observed under a scanning electron microscope.
FIG. 3 shows a photograph showing in vivo pathogenicity of sesame leaf leaves by treating the sesame leaves with spores of sesame leaf rust fungus grown in a solid medium. A: control group (spore-free group), B: spore treated group of rust fungus collected from agar plates.

In order to achieve the object of the present invention, the present invention is ammonium phosphate (NH ₄ H ₂ PO ₄ ), boric acid (H ₃ BO ₃ ), calcium chloride (CaCl ₂ ), copper sulfate pentahydrate (CuSO _{4 ~} 5H ₂ O), ethylene tetraacetic acid disodium dihydrate _{(Na 2 EDTA? 2H 2 O} ), iron sulfate heptahydrate _{(FeSO 4? 7H 2 O)} , magnesium sulfate (MgSO _4), manganese sulfate monohydrate _{(MnSO 4? H 2 O)} , potassium iodide (KI), potassium nitrate (KNO _3), zinc sulfate heptahydrate _{(ZnSO 4? 7H 2 O)} , molybdenum sodium dihydrate oxide _{(Na 2 MoO 4? 2H 2} O), agar and the number of sesame leaf comprising a cross Provides an artificial medium composition for rust mildew pure culture.

The sesame leaf rust fungus may be Coleosporium plectranthi , etc., but is not limited thereto.

The medium composition is preferably pH 5.0 ~ 7.0, more preferably pH 5.7 ~ 5.8. In addition, since the medium composition should be treated aseptically for the pure culture of sesame leaf rust fungus such as cholose porium plectranti, it is preferable to perform autoclaving before use.

In the medium composition, the concentration of ammonium phosphate (NH ₄ H ₂ PO ₄ ) may be 0.1 ~ 0.5 g / L, preferably 0.3 g / L. The concentration of boric acid (H ₃ BO ₃ ) may be 0.003 to 0.007 g / L, preferably 0.005 g / L. The concentration of calcium chloride (CaCl ₂ ) may be 0.1 ~ 0.2 g / L, preferably 0.151 g / L. The concentration of the copper sulfate pentahydrate (CuSO ₄ ~ 5H ₂ O) may be 0.0003 to 0.0007 g / L, preferably 0.0005 g / L. The concentration of ethylenediaminetetraacetic acid disodium dihydrate (Na ₂ EDTA? 2H ₂ O) may be 0.01 to 0.03 g / L, but preferably 0.02 g / L. The concentration of the iron sulfate heptahydrate (FeSO ₄ ~ 7H ₂ O) may be 0.005 ~ 0.03 g / L, preferably 0.015 g / L. The concentration of magnesium sulfate (MgSO ₄ ) may be 0.1 to 0.3 g / L, but preferably 0.195 g / L. The concentration of the manganese sulfate monohydrate (MnSO ₄ ? H ₂ O) may be 0.0005 to 0.002 g / L, preferably 0.001 g / L. The concentration of the potassium iodide (KI) may be 0.0005 to 0.002 g / L, preferably 0.001 g / L. The concentration of potassium nitrate (KNO ₃ ) may be 1 to 4 g / L, preferably 2.5 g / L. The concentration of zinc sulfate heptahydrate (ZnSO ₄ ~ 7H ₂ O) may be 0.0005 ~ 0.002 g / L, preferably 0.001 g / L. The concentration of the molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ ~ 2H ₂ O) may be 0.0005 ~ 0.002 g / L, preferably 0.001 g / L. The concentration of the agar may be 8 ~ 12 g / L, preferably 10 g / L. The concentration of sucrose may be 25 to 35 g / L, preferably 30 g / L.

Therefore, the present invention more preferably

Ammonium phosphate (NH ₄ H ₂ PO ₄ ) 0.1 to 0.5 g / L, boric acid (H ₃ BO ₃ ) 0.003 to 0.007 g / L, calcium chloride (CaCl ₂ ) 0.1 to 0.2 g / L, copper sulfate pentahydrate (CuSO ₄ ? 5H ₂ O) 0.0003 to 0.0007 g / L, ethylenediaminetetraacetic acid disodium dihydrate (Na ₂ EDTA? 2H ₂ O) 0.01 to 0.03 g / L, iron sulfate heptahydrate (FeSO ₄ ? 7H ₂ O) 0.005 to 0.03 g / L, magnesium sulfate (MgSO ₄ ) 0.1 to 0.3 g / L, manganese sulfate monohydrate (MnSO ₄ ? H ₂ O) 0.0005 to 0.002 g / L, potassium iodide (KI) 0.0005 to 0.002 g / L, potassium nitrate (KNO ₃ ) 1 to 4 g / L, zinc sulfate heptahydrate (ZnSO ₄ ? 7H ₂ O) 0.0005 to 0.002 g / L, molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ ? 2H ₂ O) 0.0005 to 0.002 g / It provides an artificial medium composition for sesame leaf rust fungus pure culture, such as Coleosporium plectranthi containing L, agar 8 ~ 12 g / L and sucrose 25 ~ 35 g / L,

Most preferably

0.3 g / L ammonium phosphate (NH ₄ H ₂ PO ₄ ), 0.005 g / L boric acid (H ₃ BO ₃ ), 0.151 g / L calcium chloride (CaCl ₂ ), copper sulfate pentahydrate (CuSO ₄ ? 5H ₂ O) 0.0005 g / L, ethylenediaminetetraacetic acid disodium dihydrate _{(Na 2 EDTA? 2H 2 O} ) 0.02 g / L, iron sulfate heptahydrate _{(FeSO 4? 7H 2 O)} 0.015 g / L, magnesium sulfate (MgSO ₄₎ 0.195 g / L, manganese sulfate monohydrate (MnSO ₄ ? H ₂ O) 0.001 g / L, potassium iodide (KI) 0.001 g / L, potassium nitrate (KNO ₃ ) 2.5 g / L, zinc sulfate heptahydrate (ZnSO ₄ ? 7H ₂ O) Choleosporium plantate containing 0.001 g / L, molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ -2H ₂ O) 0.001 g / L, agar 10 g / L and sucrose 30 g / L It provides an artificial medium composition for pure culture of sesame leaf rust fungus such as Coleosporium plectranthi ).

The present invention also provides a solid medium prepared using the artificial medium composition.

In addition, the present invention is inoculated with sesame leaf rust fungus spores, such as cholose porium Plectranty on the solid medium and cholose porium plant including the step of culturing at 22 ~ 27 ℃ in the inoculated solid medium Provides a pure culture method of sesame leaf rust fungus. The culture temperature of sesame leaf rust fungus such as cholose porium plectranti in the solid medium may be 22 ~ 27 ℃, preferably 23 ~ 25 ℃. In addition, the culture is preferably carried out in the dark conditions, it can be used by subcultured at weekly intervals, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Collection of Sesame Leaf Rust

Sesame Rust was collected in July 2010 in Gyeongsan, Korea.

Culture medium

Ammonium phosphate _{(NH 4 H 2 PO 4)} 0.3 g, of boric acid _{(H 3 BO 3) 0.005 g} , calcium chloride (CaCl ₂₎ 0.151 g, copper sulfate pentahydrate _{(CuSO 4? 5H 2 O)} 0.0005 g, ethylenediaminetetraacetic acid is 0.02 g of sodium dihydrate (Na ₂ EDTA? 2H ₂ O), 0.015 g of iron sulfate heptahydrate (FeSO ₄ ? 7H ₂ O), 0.195 g of magnesium sulfate (MgSO ₄ ), manganese sulfate monohydrate (MnSO ₄ ? H ₂ O) ) 0.001 g, potassium iodide (KI) 0.001 g, potassium nitrate (KNO ₃₎ 2.5 g, zinc sulfate heptahydrate _{(ZnSO 4? 7H 2 O)} 0.001 g, molybdenum sodium dihydrate oxide _{(Na 2 MoO 4? 2H 2} O ) A new KS (Kang and Sharif's) medium was prepared by adding distilled water to a final volume of 1 L in 0.001 g, 10 g of Difco Bacto Agar and 30 g of sucrose. The pH of the medium was adjusted to 5.7-5.8 before autoclaving (121 ° C./15 minutes) using 1 N HCl and 1 N NaOH.

Culture condition

Plastic Petri dishes (9 cm in diameter) were filled with 25-30 mL of KS medium and sealed with Parafilm M ^™ (American National) after inoculation. Optimal temperatures for the establishment and maintenance of spore germination and vegetative mycelium were determined by incubating Petri dishes at 18, 25 and 29 ° C. A total of nine Petri dishes (3 Petri dishes × temperature) were used. To minimize contamination, spore samples were inoculated into solid medium using sterile sorbet or small sterile spatula. Inoculated solid medium was examined daily by visual and stereoscopic microscopy in the first week to assess mold development and detect and remove any contamination with sterile surgical mass. Colonies were maintained in dark conditions and passaged at weekly intervals by transferring a portion of about 3-5 mm ² to fresh growth medium. The experiment was performed for 3 months.

Microscopic examination

For light microscopy, sesame leaf rust spore derived minicolon was observed every two days. Agar blocks containing mycelium were removed and transferred to slides, pressed with cover slips, and observed under an optical microscope using an oil-emulsion. The shape and surface structure of the spores were examined by scanning electron microscopy (SEM) to support the optical microscopic properties of the fungus. For scanning electron microscopy, the spores were sprayed onto a specimen holder with double-sided adhesive tape, coated with gold using a sputter coater (Hitachi E-1010 Ion Sputter, Japan), and then scanning electron microscopy ( Hitachi S-3000N, Japan) at 15 kV.

In vivo Pathogenicity Analysis of Perilla Leaf Leaves

For in vivo pathogenicity analysis, freshly grown fungi in solid medium were collected from the plates and mixed with distilled water to prepare spore suspensions. The spore suspension of sesame leaves The leaves were sprayed on and kept under greenhouse conditions of 24 ± 1 ° C. The optimum relative humidity for greenhouse growing sesame leaves was maintained at 65-75%. The control was sprayed only with distilled water.

Example 1 Sesame Leaf Rust Mold Coleosporium plectranthi Growth according to incubation temperature

Initial growth of mycelium was somewhat slow during initial incubation (3-6 days) at all three culture temperatures at 18, 25 and 29 ° C., especially at 18 and 29 ° C. Accurate observations and comparisons during the first 10 days of incubation indicated that the optimum temperature for pure culture of rust fungus was about 25 ° C. Therefore, in further experiments, cultures in all solid prostheses were incubated at 24 ± 1 ° C.

Example 2: Physical and Morphological Characteristics of Sesame Leaf Rust Mold

Once the initial colonies were more than 3 mm, they were transferred to a new solid medium. The passage culture colonies grew at lower rates with downy and loose mycelium, most of which turned brown within 3-4 weeks (FIG. 1).

Optical microscopic characteristics of urediospore and uredinium are shown in FIG. 2A. The follicles are single cells, dinuclear, and spines on the outer wall. And yellow or orange when immature. 2B shows the results of scanning electron microscopy of the spores and spores regression.

Example 3: In Vivo Pathogenicity of Perilla Leaf Rust Fungus for Perilla Leaf Leaves

In vivo pathogenicity was determined by the presence or absence of disease zone in the sesame leaf tested. The rust was observed in the leaves of the treatment group 14-15 days after inoculation of the sesame leaf rust fungus spores collected by incubation in the solid medium (FIG. 3B), whereas the control leaves (distilled water treatment) did not show symptoms of rust (FIG. 3A).

Claims (6)

Ammonium phosphate _{(NH 4 H 2 PO 4)} , boric acid (H ₃ BO _3), calcium chloride (CaCl _2), copper sulfate pentahydrate _{(CuSO 4? 5H 2 O)} , ethylenediaminetetraacetic acid disodium dihydrate (Na ₂ EDTA? 2H ₂ O), iron sulfate heptahydrate _{(FeSO 4? 7H 2 O)} , magnesium sulfate (MgSO _4), manganese sulfate monohydrate _{(MnSO 4? H 2 O)} , potassium iodide (KI), potassium nitrate (KNO ₃₎ The artificial medium composition for pure culture of sesame leaf rust fungus comprising zinc sulfate heptahydrate (ZnSO ₄ ~ 7H ₂ O), molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ ~ 2H ₂ O), agar and sucrose. The artificial medium composition of claim 1, wherein the sesame leaf rust fungus is Coleosporium plectranthi . The artificial medium composition of claim 1, wherein the artificial medium composition has a pH of 5.0 to 7.0. According to claim 1, ammonium phosphate (NH ₄ H ₂ PO ₄ ) 0.1 ~ 0.5 g / L, boric acid (H ₃ BO ₃ ) 0.003 ~ 0.007 g / L, calcium chloride (CaCl ₂ ) 0.1 ~ 0.2 g / L, copper sulfate pentahydrate _{(CuSO 4? 5H 2 O)} 0.0003 ~ 0.0007 g / L, ethylenediaminetetraacetic acid disodium dihydrate _{(Na 2 EDTA? 2H 2 O} ) 0.01 ~ 0.03 g / L, iron sulfate heptahydrate (FeSO _4? 7H ₂ O) 0.005 to 0.03 g / L, magnesium sulfate (MgSO ₄ ) 0.1 to 0.3 g / L, manganese sulfate monohydrate (MnSO ₄ ? H ₂ O) 0.0005 to 0.002 g / L, potassium iodide (KI) 0.0005 to 0.002 g / L, potassium nitrate (KNO ₃ ) 1 to 4 g / L, zinc sulfate heptahydrate (ZnSO ₄ ? 7H ₂ O) 0.0005 to 0.002 g / L, molybdenum oxide sodium dihydrate (Na ₂ MoO ₄ ? 2H ₂ O ) 0.0005 ~ 0.002 g / L, agar 8 ~ 12 g / L and sucrose leaf rust fungus artificial medium composition containing 25 to 35 g / L pure culture. Solid medium prepared using the artificial medium composition of any one of claims 1 to 4. A method of pure culture of sesame leaf rust fungus comprising the step of inoculating sesame leaf rust fungus spores into the solid medium of claim 5 and culturing at 22 ~ 27 ℃ in the inoculated solid medium.

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