KR20180054000A - Selective medium for isolation of Botyrtis cinerea and Use Thereof - Google Patents

Abstract

The present invention relates to a selective medium capable of selectively isolating Botrytis cinerea, a production method thereof, and a use thereof. More specifically, the present invention relates to a selective medium capable of selectively isolating and growing Botrytis cinerea which causes gray mold rot, and a method for discriminating resistance to germicides using the same.

Description

Selective Medium for Isolation of Botrytis cinerea and Use Thereof

The present invention relates to a selective medium of Vortis crisne, a method for producing the same, and uses thereof.

KOREA INSAM is an important natural resource of Korea. It is a representative herb medicine of Korea, which has been widely used for medical purposes as well as oriental medicine for mankind for many millennia. With the adverse effects of synthetic medicines and the overcoming of intractable diseases, western medicine is showing its limit, and the value of ginseng and other herbal medicines and natural products is newly emerging. It is also reported that ginseng prevents cancer, inhibits aging, protects the liver, strengthens fatigue and brain function, and recently prevents environmental hormone damage. Ginseng, the first of all herbal medicines, is a plant that grows in an extremely limited environment. It has a longer growth period than other crops, and the cultivation method is also special and difficult.

Korea is a ginseng suzerain producing climatic ginseng, which is excellent in its medicinal effect due to its natural climate and cultivation area. However, the interest of many countries in the ideal condition of ginseng as a health food or health care drug has increased, Non-producing countries, on the other hand, are actively promoting policies that encourage research and production of ginseng. In Korea, China, Japan, the United States, and some parts of Europe, ginseng has been cultivated in Korea, China, Japan, Taiwan and Southeast Asia. Is expanding. However, the international competitiveness of Korean ginseng has weakened in terms of price. In order to enhance international competitiveness, it is necessary to study the development of cultivation technology to improve the quality of ginseng as a raw material. Especially, the most urgent problems in cultivation of ginseng in recent years are the absolute lack of proper place, high incidence due to worsening of soil conditions in the country, and pesticide residue due to increase of pesticide usage. There is an urgent need for intensive research on On the other hand, the control of gray mold disease in ginseng cultivation has become an important problem from low to high age. In the case of seedling ginseng, the growth of the primordial gland occurs in the upper part of the body, resulting in an increase in the yield of the ginseng. Resource Research Institute, 2005). Symptoms caused by gray mold, which occurs frequently in ginseng cultivation, occur at all growth stages and rapidly progress to the primordia or striae, causing decay of roots, formation of a malignant lesion on the leaves, . This pathogen is in the form of a fungus or Sclerotium, which in winter grows in diseased plants or soil and spreads mainly by rain and wind. The lesion area begins with brown spots and progresses gradually to enlarged lesions of broad gray, grayish brown color and dense gray cysts. It is one of the major diseases of ginseng (ginseng cultivation, standard farming manual p 105, Rural Development Administration, 2000). Actually, the causative agent of gray mold is Botrytis cinerea which is the causative agent of ginseng roots rot, and it is more problematic because of the easy occurrence of anthrax and flaccid disease in damaged ginseng pavement. It has been reported that after March, it started to cause rotting of the primordial gland, and damages stem and leaf of ginseng until October, and is known to cause corruption during storage of ginseng after harvest. Recent climate change has increased the damage of root rot caused by the occurrence of gray mold of Zygosha japonica and it has been reported that up to 90% This disease control method has been applied to physical control to treat diseased ginseng and leaves on the ground and to disinfect and clean the packaging, and to use pesticides such as polyoxine hydrating agent, pyrimethanil liquid wetting agent, imidotidine tris lysylate + polyoxine hydrating agent Chemical control is being carried out. However, it can be seen that the above-mentioned control method is difficult to obtain a clear effect, and that a large number of drug-resistant strains are produced, and pesticides which can be controlled can be reduced. In addition, since it is a multi-pathogenic pathogen with wide host range, the occurrence of pesticide residues and drug resistance is reported when controlling with a single-line drug, and it is necessary to develop an effective control method (Korean Society of Plant Pathology, Lee et al., P 170-176 , 2007, The Korean Society of Plant Pathology). Therefore, in order to effectively control the gray mold, it is considered that the spore propagating through the scattering is periodically investigated with the spore scattering amount to identify the disease occurrence and to select the optimum pesticide, and it is useful to use the selective medium to investigate the spore scattering amount . To date, the use of selective medium to control gray mold has not been carried out in the research facilities, so it is difficult for farmers to use it directly due to lack of understanding. Especially, it is not easy for farmers to have a variety of reagents and apparatuses for the preparation of selective media containing various reagents. Therefore, when the selective medium is prepared and supplied to the farmers, it can be effectively used for the prediction and control of the gray mold pathogens.

However, the above-mentioned control method is difficult to obtain a clear effect, and soil-infectious pathogens such as ginseng root rot disease cause a disease by forming a thick spore in the soil, so that it is almost impossible to control. The use of soil fumigants has the potential to dominate the pathogen in five years due to the destruction of the soil ecosystem. In particular, the major soil diseases of ginseng are infected at various temperature ranges, so once infected, the disease progresses slowly but it is damaged throughout the year. Therefore, for the effective control of soil disease, biological control of ginseng disease is considered to be the most appropriate method for the control of ginseng disease, which is a continuous and environmentally stable disease control effect due to the characteristics of ginseng cultivation. In this regard, Cylindrocarpon The identification and antagonism of Streptomyces species that exhibit pathway resistance to destructans has been reported

So far, a method of biological control using antagonistic microorganisms has been used as a method to control ginseng root rot disease, and in particular, a chemical control method using a soil fumigant such as a sand theory, a rosemide, and a rice paddy cultivation method The method is not systematized. In particular, studies on biological control of ginseng soils have been conducted mainly on the selection of antagonistic microorganisms and on the effect of the treatment of pesticides. However, there is a lack of understanding of various rhizospheric soil environments, the antimicrobial activity of the selected strain against the target pathogen is not high, The antimicrobial activity of the ginseng root rot disease is not observed due to the inferiority of the bacterial fixing ability in the soil or the seedling field due to insufficient method or treatment method.

Accordingly, the present inventors have made efforts to develop a medium having high selectivity for pathogenic bacteria causing ginseng gray mold, root rot, and root rot, and as a result, developed a selective medium having high selectivity for gray mold pathogens, Alterna appear pathogens that cause disease Leah Alter (Alternaria alternata), conductance appeared throw Carphone Destruction (Cylindrocarpon destructans), as well as oxy-Fujairah Leeum's forum (Fusarium oxysporum), Fusarium small Lani (Fusarium solani , Pythium ultimum, Rhizoctonia solani), switch Klee Loti Nias Klee in and having a high antibacterial activity against a variety of pathogens, including thio room (Sclerotinia sclerotiorum) confirm the effect of selectively grow well only Botrytis cine Ria mycelium and spore germination (Botrytis cinerea) And completed the present invention.

(A) heating a solution containing reagent in 1 L of distilled water without sterilization and then cooling; And (b) a method for preparing a Botrytis cinerea selective medium comprising the step of adding azoxystrobin to the solution.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

(A) heating a solution containing reagent in 1 L of distilled water without sterilization and then cooling; And (b) further adding azoxystrobin to said solution.

(Botrytis cinerea) selective medium.

The reagent used in the step (a) may be selected from the group consisting of 0.1 to 20 g of Detrose, 0.01 to 4 g of potato starch, 0.005 to 2 g of chloramphenicol, 0.001 to 0.2 g of pentachloronitrobenzene (PCNB) From 0.0025 to 0.5 g of rose bangal, from 0.25 to 50 g of tannic acid and from 10 to 200 g of agar.

In (b), the concentration of azoxystrobin may be 0.01 ppm to 10000 ppm.

The Botrytis cinerea may be causing a gray mold disease.

In one embodiment of the present invention, a selective medium of Botrytis cinerea prepared according to the above method is provided.

The selective medium was prepared by adding 0.1 to 20 g of Detrose, 0.01 to 4 g of potato starch, 0.005 to 2 g of chloramphenicol, 0.001 to 0.2 g of pentachloronitrobenzene (PCNB) 0.0025 to 0.5 g of rose bangal, 0.25 to 50 g of tannic acid and 10 to 200 g of agar,

And may further comprise Azoxystrobin.

The concentration of the azoxystrobin may be 0.01 ppm to 10000 ppm.

The selective medium may be pH 4 to pH 8.

The culture for separating Botrytis cinerea from the selective medium may be carried out at a temperature of from 15 캜 to 30 캜.

The selection medium and Leah Alter Alter appear (Alternaria alternata), conductance appeared throw Carphone Destruction (Cylindrocarpon destructans), Fujairah Solarium Oxy's forum (Fusarium oxysporum), Fusarium small Lani (Fusarium solani , Pythium ultimum, Rhizoctonia solani , and Sclerotinia sclerotiorum. The antimicrobial activity of the antimicrobial agent of the present invention may be at least one kind selected from the group consisting of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum.

In another embodiment of the present invention, there is provided a method for producing a microorganism which comprises: (a) heating a selective medium of the present invention without sterilization and then cooling; (b) preparing a pharmaceutical medium by adding a sterilizing agent to the selective medium after heating; (c) collecting the spore after placing the medicament medium in the center of the ginseng package; And (d) judging that Botrytis cinerea germinates when the color of the pharmaceutical medium is brown.

The present invention provides a method for determining the fungicide resistance of a gray mold pathogen.

In (b), the fungicide may be selected from the group consisting of pyrimethanil, iminoctadine tris (albesilate), polyoxin B, fluquinconazole, fludioxonil, boscalid, prochloraz manganese complex, and the like.

The sterilizing agent may be contained in a concentration of 0.01 g to 10 g per liter of selective medium.

The gray mold fungus selective medium of the present invention selectively permits the growth of mycelial growth and spore germination of Botrytis cinerea, a causative agent of gray mold, and inhibits mycelial growth and spore germination of other plant pathogens. Have the same antimicrobial activity and have better antimicrobial activity than the previously reported BSM medium. The selection of suitable fungicide and control method for gray mold fungus is effective for the farmers and various prisoners by evaluating the isolation of gray mold fungus, the spores scattering rate and the resistance to fungicide in a wide range of crops including ginseng using the selective medium.

FIG. 1 shows the characteristics of the selective medium for the separation of the gray mold pathogens developed in the present invention.
FIG. 2 shows the results of the mycelial growth of plant pathogenic bacteria in the developed selective medium and the previously reported BSM medium and PDA medium, which is a fungal culture medium.
FIG. 3 shows the result of confirming the mycelial growth characteristics of plant pathogenic bacteria in the selective medium developed in the present invention.
FIG. 4 is a graph showing the mycelial growth characteristics of the gray mold pathogens according to the concentration of Azoxystrobin in the selective medium developed in the present invention.
FIG. 5 is a graph showing the effect of inhibiting mycelial growth of plant pathogenic bacteria according to the concentrations of Azoxystrobin 3 days after inoculation of plant pathogenic bacteria in the selective medium developed in the present invention.
FIG. 6 shows the results of confirming the effect of inhibiting mycelial growth of plant pathogens according to the concentrations of Azoxystrobin 7 days after plant propagation in the selective medium developed in the present invention.
FIG. 7 shows collected spores of gray mold fungi scattered in ginseng packaging using the selective medium developed in the present invention.
FIG. 8 shows a selective medium developed in the present invention in which a batch is installed to collect gray mold pathogens in ginseng packaging.
FIG. 9 shows the results of evaluation of fungicide resistance of ginseng gray mold pathogens using the selective medium developed in the present invention.

As described above, a selective medium capable of isolating Botrytis cinerea, a pathogenic bacterium causing gray mold, has not been developed. In addition, a technique for determining spore scattering and drug resistance of pathogens has been used, but it takes more than two months to evaluate the drug resistance by isolating pathogens from plants. In addition to the gray mold pathogens, the previously reported selective medium enables the growth of other pathogens, and thus the activity of the selective medium is deteriorated.

Accordingly, the present inventors sought to solve the above-mentioned problem by providing a selective medium capable of selectively isolating Botrytis cinerea. In addition, the selective medium of the present invention can be easily prepared through heating without sterilization.

The term " Botrytis cenerea "in the present specification is a pathogenic bacterium causing gray mold fungus, root rot, and rot fungus disease of ginseng. By selectively isolating and growing the pathogenic fungus through the selective medium of the present invention, And inhibit the growth of mycelial growth of mycelia and accelerate spore germination.

Hereinafter, the present invention will be described in detail.

(A) heating a solution containing reagent in 1 L of distilled water without sterilization and then cooling; And (b) further adding azoxystrobin to the solution. The present invention also provides a method for preparing a selected medium for Botrytis cinerea.

The present invention is characterized in that a solution containing reagent in distilled water is heated without sterilization in order to overcome the problem that the efficiency of selective separation with other strains is poor in the conventional selective medium.

 In the step (a), the solution can be easily prepared by omitting the sterilization process, and thus, the mycelial growth of Botrytis cinerea and mycelial growth of other pathogens, By effectively inhibiting spore germination, Botrytis cinerea can be selectively isolated.

The heating temperature is preferably 80 ° C to 120 ° C, more preferably 100 ° C to 110 ° C, but is not limited thereto. At this time, when the heating temperature is lower than the above range, there is a problem that the agar does not melt. When the heating temperature exceeds the above range, there is a problem that the medium overflows and flows.

Further, it is preferable to cool the solution at a temperature of 40 ° C to 60 ° C after heating, and it is preferable to cool at a temperature of 50 ° C to 55 ° C, but is not limited thereto. At this time, when the cooling temperature is lower than the above range, there is a problem that the culture medium is hardened, and when the cooling temperature exceeds the above range, the activity of the bactericide is lowered.

In (b), the concentration of azoxystrobin may be 0.01 ppm to 10000 ppm. In one embodiment of the present invention, in order to confirm the degree of activity according to the concentration of azoxystrobin added to the selective medium of the present invention, the concentration of azoxystrobin was adjusted to 200, 500 and 1000 ppm, respectively, The mycelial growth was investigated. As a result, as shown in FIG. 5, the difference in growth between BSM medium and other pathogens was not confirmed after 3 days of inoculation, but azosistrobin was added at 200, 500 and 1000 ppm It was confirmed that the growth of Botrytis cinerea was significantly superior to the growth of other strains in one selective medium.

Further, as shown in Fig. 6, growth of Botrytis cinerea was excellent regardless of the concentration of azoxystrobin in the medium supplemented with 200, 500 and 1000 ppm of azoxystrobin even after 7 days of inoculation, It was confirmed that the mycelial growth of other pathogenic bacteria was significantly inhibited when compared with the medium.

That is, the selective medium of the present invention is excellent in growth of Botrytis cinerea causing gray mold regardless of the concentration of azoxystrobin contained in the selective medium. Therefore, Spore collection and selective isolation of Botrytis cinerea is possible.

The present invention also provides a selective medium of Botrytis cinerea prepared according to the above method.

In the case of the selective medium prepared according to the above method, Botrytis cinerea, a pathogenic bacterium causing gray mold, can be selectively isolated and grown.

The selective medium is preferably pH 4 to pH 8, more preferably pH 4.5 to pH 7.5, but is not limited thereto. When the pH is less than the above range, there is a problem in that the medium is not hardened. When the pH exceeds the above range, the problem is that the brown color formed around the growth medium of Botrytis cinerea grows on the selective medium have.

The culture for isolating Botrytis cinerea in the selective medium is preferably performed at a temperature of 10 to 30 DEG C, more preferably at a temperature of 15 to 25 DEG C, It does not. At this time, when the incubation temperature is lower than the above range, there is a problem that the growth of Botrytis cinerea is delayed, and when the incubation temperature exceeds the above range, the growth of Botrytis cinerea There is a problem that growth is limited.

The selection medium and Leah Alter Alter appear (Alternaria alternata), conductance appeared throw Carphone Destruction (Cylindrocarpon destructans), Fujairah Solarium Oxy's forum (Fusarium oxysporum), Fusarium small Lani (Fusarium solani , Pythium ultimum, Rhizoctonia solani , and Sclerotinia sclerotiorum. The antimicrobial activity of the antimicrobial agent of the present invention may be at least one kind selected from the group consisting of Sclerotinia sclerotiorum and Sclerotinia sclerotiorum. Specifically, in one embodiment of the present invention, in order to evaluate the antimicrobial activity of the selective medium, BSM medium, and PDA medium of the present invention, Botrytis cinerea, a gray mold fungus, and Alteraria altaeri, Alternaria alternata, Cylindrocarpon destructans , Fusarium < RTI ID = 0.0 > the degree of inhibition of the mycelial growth of Fusarium solani , Pythium ultimum, Rhizoctonia solani and Sclerotinia sclerotiorum was determined to be Respectively. As a result, it was confirmed that the selective medium of the present invention enables mycelial growth of Botrytis cinerea and inhibits growth of other pathogens.

In another embodiment of the present invention, the effect of inhibiting mycelial growth of Botrytis cinerea, a gray mold fungus pathogen, and seven plant pathogens, using the selective medium of the present invention, , The growth of other strains except Botrytis cinerea was very low in the selective medium after one week of inoculation and the mycelial growth of Botrytis cinerea resulted in the growth of mycelium Was changed to brown color and the tannic acid in the vital selection medium was decomposed to change the color of the medium from pale pink to pale yellow.

(A) heating the selective medium of the present invention without sterilization and then cooling; (b) preparing a pharmaceutical medium by adding a sterilizing agent to the selective medium after heating; (c) collecting the spore after placing the medicament medium in the center of the ginseng package; And (d) judging that Botrytis cinerea is germinated when the color of the pharmaceutical medium is brown. The present invention also provides a method for identifying a fungicide resistance of ginseng gray mold pathogens.

In (a), the heating temperature is preferably 80 ° C to 120 ° C, more preferably 100 ° C to 110 ° C, but is not limited thereto. At this time, when the heating temperature is lower than the above range, there is a problem that the agar does not dissolve. When the heating temperature exceeds the above range, there is a problem that the medium overflows and flows.

Further, it is preferable to cool the solution at a temperature of 40 ° C to 60 ° C after heating, and it is preferable to cool at a temperature of 50 ° C to 55 ° C, but is not limited thereto. At this time, when the cooling temperature is lower than the above range, there is a problem that the culture medium is hardened, and when the cooling temperature exceeds the above range, the activity of the bactericide is lowered.

In (b), the fungicide may be selected from the group consisting of pyrimethanil, iminoctadine tris (albesilate), polyoxin B, fluquinconazole, fludioxonil, boscalid, prochloraz manganese complex, and the like.

The sterilizing agent is preferably contained in a concentration of 0.05 g to 2 g in 1 L of the selective medium according to the safe use standard of the pesticide, more preferably 0.5 g to 1 g, but is not limited thereto. At this time, when the concentration of the bactericide contained in 1 L of the selective medium is less than the above range, there is a problem that the resistance to fungicide can not be evaluated, and when the concentration of the bactericide exceeds the above range, the resistance to fungicide can not be evaluated properly.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[ Example ]

Example  One. A gray mold pathogen Optional badge  Composition and recipe

A medium capable of selectively isolating Botrytis cinerea, a gray mold pathogen, was prepared, and the composition of the selective medium was shown in Table 1 below.

2 g of Dextrose, 0.4 g of potato starch, 0.2 g of chlorampenicol, 0.02 g of pentachloronitrobenzene (PCNB), 0.05 g of rose bangal, 5 g of tannic acid and 20 g of agar were added to 1 L of distilled water and boiled while mixing. The mixture was cooled to 50 ° C and 200 mg of azoxystorbin Dishes were dispensed in a volume of 25 to 30 ml, and the medium was fixed on a sterile stand for 30 minutes to prepare a medium. The prepared selective medium was characterized by a light pink color (Fig. 1).

ingredient Capacity (1 L) Dextrose 2g Potato starch 0.4 g Potassium cloride 0.1 g Chloramphenicol 0.2 g Pentachloronitrobenzene 0.02 g Rose barbecue 0.05 g Tannic acid 5g azoxystrobin 0.2 g Agar 20g

Example  2. Gray mold Selective  Activity evaluation

In order to evaluate the activity of the selective medium developed in Example 1, Botrytis cinerea, a plant belonging to the gray mold, and Alternaria alternata , Cylindrocarpon destructans , Fusarium < RTI ID = 0.0 > oxysporum , Fusarium solani , Pythium ultimum, Rhizoctonia solani , and Sclerotinia sclerotiorum are disclosed in selection media Were investigated. The mycelial growth of pathogens was compared with those of BSM medium and PDA medium, which were previously reported by Edwards and Sedden (2001). Each pathogen was cultured in PDA medium for 1 week and pierced using a sterilized 6mm cork borer to make hyphae. The hyphae of pathogenic bacteria were placed on the center of the selective medium, and the activity of the selective medium was evaluated by incubating the medium at 20 ° C for 1 week.

The mycelial growth of the gray mold pathogens was good in the previously reported BSM medium (Edwards and Sedden, 2001), but mycelial growth of Cylindrocapone dis- tortense, Fusarium saurani and Fusarium oxysporum was relatively good and the selectivity was poor Respectively. As a result of evaluating the activity of the selected selective medium, the growth of other bacteria except for the gray mold pathogens was very low and the mycelial growth of the gray mold pathogens was excellent in the selective medium after 1 week of inoculation (FIG. 2). In addition, the hyphae of mycelium changed to brown with the growth of hyphae of the gray mold pathogens, and the color of the medium changed from light pink to pale yellow by decomposing the tannic acid in the medium. In addition to the published pathogens, it was confirmed that only the mycelium of the gray mold pathogens selectively grew well in the developed selective medium (FIG. 3).

Thus, in the present invention, the selected medium was named as a gray mold pathogen selection medium.

Example  3. Azoxystrobin  Different in concentration Selective  Activity evaluation

Botrytis , a ginseng gray mold pathogen, cinere a pathogen with 7 species of ginseng and Leah Alter Alter appear (Alternaria alternata), as well as draw Carphone Destruction capacitance (Cylindrocarpon destructans) appeared, Fujairah Solarium Oxy's forum (Fusarium oxysporum), Fusarium small Lani (Fusarium solani , Pythium ultimum, Rhizoctonia solani ) and Sclerotinia sclerotiorum were investigated. Each pathogen was cultured on PDA medium for 1 week and pierced using a sterilized 6 mm corcovorer to produce hyphae. Each mycelial slice was placed at the center of the selective medium containing 200, 500, and 1000 ppm of azoxystrobin, and the activity was evaluated by incubating at 20 ° C for 1 week. The mycelial growth of eight selected plant pathogens was investigated in BSM medium and PDA medium, which were developed by Edwards and Sedden (2001).

After 3 days of inoculation on the selective medium, it was not possible to distinguish the other pathogens from the gray mold fungi in the BSM medium. However, in the medium supplemented with 200, 500, and 1000 ppm of azoxystrobin, It was confirmed that the growth of gray mold pathogens was fast (Fig. 5).

In addition, in the results after 7 days of inoculation, the growth of gray mold fungi was excellent regardless of the concentration of azoxystrobin in the medium containing 200, 500 and 1000 ppm of azoxystrobin (Fig. 4) (Fig. 6). ≪ tb > < TABLE > In the developed selective medium, the gray mold was dark brown and easily distinguishable from other plant pathogens, and the growth of the gray mold pathogens was easier to visualize than the previously reported BSM medium (Edwards and Sedden, 2001) .

Example  4. Select your badge  Used Ginseng gray mold  Spore Scattered amount  Research

In order to investigate the distribution of the gray mold fungus pathogens using the selective medium developed in Example 1, spores of the gray mold pathogens were collected from the ginseng 2 and 5 year old ginsengs located in the National Institute of Horticultural Science. Plants were placed in the grooves of 1/3, 1/2, and 3/3 of the ginseng packaging from 2 pm to 6 pm, with four selected media on top, and the lid was left open for 4 hours. After 4 hours, the selective medium was collected and cultured in a 20 ° C incubator for 3 to 7 days, and the spores of the collected gray mold pathogens were examined.

In the BSM medium reported by Edwards and Sedden (2001), it was found that the germs such as Alternaira, Fusarium , Penicillum and the like were germinated easily in the medium in addition to the gray mold pathogens. However, in the developed selective medium, (Fig. 7). As shown in Fig.

As a result of investigating the concentration of the ginseng gray mold (porosity / 90 mm plate) of the ginseng gray mold pathogens, there was no significant difference in the collected conidia when compared with the BSM medium and the selected medium, It was confirmed that a lot of spores were collected from the 5-year-old pavement than in the pavement, and the results are shown in Table 2 below.

Packing Year BSM Optional badge A 2 4 3 B 2 3 2 C 2 5 4 D 5 31 31 E 5 41 48

Example  5. Select your badge  Utilized Ginseng gray mold  Fungicide resistance determination

The selected medium was boiled without sterilization and then cooled to 50 DEG C. Nine kinds of bactericides used in the farm were selected and added with each bactericide according to the use concentration according to the safe use standard of the pesticide as shown in Table 3, 20 ml each was dispensed to adjust the drug medium.

Three ginseng pavements were selected from Chungju, Gosung, Goesan, and Jeongpyeong areas in Chungbuk province. A plastic box was placed in the center of the ginseng pavement, and two medicinal herbs were placed on top of the plastic box. The spores were collected by opening the lid for 4 hours (FIG. 8). The culture was incubated at 20 ℃ for 1 week.

Item name Brand name Dilution volume / 20L
(Dilution factor) Active ingredient Ingredients Addition amount / 1 L (selective medium) Pyrimethanil liquid wettable powder Mitos 20g (1,000 times) 37% pyrimethanil 1g ≪ RTI ID = 0.0 > already < / RTI > Belkut 20g (1,000 times) 40% iminoctadine tris 1g Imidazolidinone < / RTI > < RTI ID = 0.0 > Red Toma 20g (1,000 times) 20%
10% iminoctadine trispolyoxin B 1g Fluqueenquinazoline / pyrimethanil liquid wettable powder Gold Mori 20ml (1,000 times) 5%
30% fluquinconazolepyrimethanil 1 ml Penhexamide liquid wettable powder Telldo 20ml (1,000 times) 42% fenhexamid 1 ml Fluodioxonil liquid wettable powder Sapphire 10ml (2,000 times) 20% fludioxonil 0.5 ml Boss Calid granular wettable powder Kantus 13.3g 49.3% boscalid 0.67 g Polyoxine wettable powder Eastern polyoxine 20g (1,000 times) 10% polyoxin B 1g Prochloraz manganese hydrate Spoogon 10g (2,000 times) 50% prochloraz manganese complex 0.5 g

As a result of investigating the resistance of the germicide used for the control of gray mold in Chungbuk province, Gyeongbuk province, by using selective medium, it was found that after 5 days of spore collection, the spore germination point of the gray mold pathogenic bacteria was around the gray hyphae It was confirmed to have a brown color (Fig. 9). Most spores of other pathogens were inhibited, and some spores did not have a brown band at the germination point.

The evaluation of bactericidal resistance strains was confirmed by comparing the number of spores of germinated fungal pathogens germinated in a selective medium without the addition of bactericides. As a result of evaluating drug resistance by region, resistant strains against polyoxin hydrate were identified in Jeongpyeong, Goesan and Chungju regions as shown in Table 4, and resistant strains against penhexamide liquid wetter were started to occur in Goesan and Chungju regions. In addition, resistant strains against fluodoxonil liquid wettable powders and boscarried granular wettable powders were identified in some areas of Goesan, Chungju, and Cheongpyeong. In all of the sites investigated, it was found that in all the areas, the pirimanil liquid wettable agent, imidoxydrine tris lysylate wettable agent, iminotadine tris lysylate + polyoxine wettability agent, fluquinonezol + pyrimethanil liquid wettable agent and prochloraz manganese wettability agent No resistant strains were identified. In case of collecting pathogens directly and analyzing the bactericides, about 2 months' time is used to evaluate the resistance of the pathogens from the pure isolation of the pathogens. However, the technology to evaluate the fungicide resistance against the gray mold pathogens using the selective medium, It is considered that the fungicide resistance of gray mold pathogens can be discriminated inside and outside. When these techniques are applied to farm households, it is expected that they will be able to effectively control disease by using the best medicine for gray mold disease.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (13)

(a) heating a solution containing reagent in 1 L of distilled water without sterilization and then cooling;
And
(b) further adding azoxystrobin to said solution.
A method for producing a selective medium of Botrytis cinerea.
The method according to claim 1,
The reagent used in the step (a) may be selected from the group consisting of 0.1 to 20 g of Detrose, 0.01 to 4 g of potato starch, 0.005 to 2 g of chloramphenicol, 0.001 to 0.2 g of pentachloronitrobenzene (PCNB) 0.0025 to 0.5 g of rose bangal, 0.25 to 50 g of tannic acid and 10 to 200 g of agar.
A method for producing a selective medium of Botrytis cinerea.
The method according to claim 1,
The concentration of azoxystrobin in (b) is 0.01 ppm to 10000 ppm.
A method for producing a selective medium of Botrytis cinerea.
The method according to claim 1,
The Botrytis cinerea is characterized by causing gray mold disease
A method for producing a selective medium of Botrytis cinerea.
A selective medium of Botrytis cinerea prepared according to the method of claim 1.
6. The method of claim 5,
The selective medium was prepared by adding 0.1 to 20 g of Detrose, 0.01 to 4 g of potato starch, 0.005 to 2 g of chloramphenicol, 0.001 to 0.2 g of pentachloronitrobenzene (PCNB) 0.0025 to 0.5 g of rose bangal, 0.25 to 50 g of tannic acid and 10 to 200 g of agar,
Characterized in that it further comprises Azoxystrobin
Selective medium for Botrytis cinerea.
The method according to claim 6,
Characterized in that the concentration of the azoxystrobin is from 0.01 ppm to 10000 ppm
Selective medium for Botrytis cinerea.
6. The method of claim 5,
Characterized in that the selective medium is pH 4 to pH 8
Selective medium for Botrytis cinerea.
In the fifth aspect,
Characterized in that the culture for isolating Botrytis cinerea in the selective medium is carried out at a temperature of 10 ° C to 30 ° C
Selective medium for Botrytis cinerea.
6. The method of claim 5,
The selection medium and Leah Alter Alter appear (Alternaria alternata), conductance appeared throw Carphone Destruction (Cylindrocarpon destructans), Fujairah Solarium Oxy's forum (Fusarium oxysporum), Fusarium small Lani (Fusarium solani , Pythium ultimum, Rhizoctonia solani , and Sclerotinia sclerotiorum. The antimicrobial agent according to any one of claims 1 to 3,
Selective medium for Botrytis cinerea.
(a) heating the selective medium of claim 5 without sterilization and then cooling;
(b) preparing a pharmaceutical medium by adding a sterilizing agent to the selective medium after heating;
(c) collecting the spore after placing the medicament medium in the center of the ginseng package; And
(d) determining that Botrytis cinerea germinates when the color of the pharmaceutical media is brown
Determination of fungicide resistance of gray mold pathogens.
12. The method of claim 11,
In (b), the fungicide may be selected from the group consisting of pyrimethanil, iminoctadine tris (albesilate), polyoxin B, fluquinconazole, fludioxonil, boscalid, and prochloraz manganese complex.
Determination of fungicide resistance of gray mold pathogens.
12. The method of claim 11,
Characterized in that the sterilizing agent is contained in a concentration of 0.01 g to 10 g per liter of selective medium
Determination of fungicide resistance of gray mold pathogens.

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