KR101385200B1 - Disease diagnosis kit including species-specific primer, and detection and disease diagnosis method for Cylindrocarpon destructans - Google Patents

Abstract

The present invention relates to ginseng root rot bacteria ( Cylindrocarpon destructans ) detection, and pathogenic diagnostic method, and more specifically, ginseng root rot bacteria including a primer set for detecting and diagnosing pathogenic bacteria, The present invention relates to a kit for detecting and diagnosing pathogenicity and a method for diagnosing, counting and pathogenicity of ginseng root rot bacteria. The present invention can be used to reduce the time and cost required to diagnose ginseng root rot bacteria and to increase the accuracy of the determination of infection, thereby reducing the risk and economic loss caused by root rot disease.

Description

Disease diagnosis kit including species-specific primer, and detection and disease diagnosis method for Cylindrocarpon destructans

The present invention relates to a method for detecting and/or diagnosing pathogenicity of ginseng root rot ( Cylindrocarpon destructans ), and more specifically, a primer set for detecting and/or diagnosing pathogenicity of ginseng root rot, ginseng root rot including the primer set It relates to a kit for detecting and diagnosing pathogenic bacteria, and a method for detecting, counting, and diagnosing pathogenicity of ginseng root rot using the primer set.

Ginseng botanically belongs to the ginseng genus of the family Araliace, and its scientific name is Panax ginseng C.A Meyer, a herbaceous plant whose roots are used for medicinal purposes.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used in Korea, China, and Japan, mainly for health promotion purposes. The active ingredients of ginseng are known to have remarkable differences depending on the type of ginseng, the root part, the year of harvest, the time of harvest, and the manufacturing method. It is known to be.

Ginseng is a substance that has the ability to recover from fatigue such as exercise ability, stress response effect, cognitive ability, and psychological stability, and it has been found in the ginseng research literature of Bahrke et al. (2000), and has a non-specific defense action against foreign substances such as infectious substances in the body. As an activating substance, it has been scientifically proven to have an immunity-strengthening effect.

Ginseng is a perennial semi-shaded sukkeuncho (宿根草), with a photosynthetic light compensation point of only 0.5klux of leaves, and it is a semi-morbid plant that only needs light of relatively low luminosity for growth, so light is the most important growth limiting factor. In addition, ginseng is a low-temperature plant whose growth is inhibited at high temperatures, and since it is vulnerable to high temperatures and high brightness, temperature is an important growth limiting factor, and the optimal growth temperature of Korean ginseng is estimated to be in the range of 18-21. Ginseng is evaluated as a plant that is difficult to cultivate because it has weak disease resistance and few effective control methods in addition to the above growth limiting factors.

The plant of ginseng is composed of roots and brain heads under the ground, stems, leaves, and flowers (fruits) on the ground. Since it is a perennial plant, sprouts emerge from the roots in the ground every spring, and the stems die every autumn. It leaves a trace of growth every year on the 頭). The growth and shape of the roots are different depending on the year of birth, and the harvest is done at the age of 4-6 years. Ginseng must be cultivated for a long time in the same field for 4 to 6 years, and after harvesting ginseng, no other crops are cultivated. It is very large and there is a high possibility of occurrence of continuous cropping disorder.

Serial disturbances include Phytophthora cactorum , Cylindrocarpon destructans , Fusarium solani , Erwinia carotovora , and Pseudomonas fluorescens fluorescens fluores. It is known to occur because pathogens act alone or in combination.

According to a recent study by the Institute of Agricultural Technology, the cause of absence from ginseng over 4 years old is root rot disease 35%, gray mold disease 32%, stem spot disease, etc., accounting for 15%, although the cause of the absence of ginseng over 4 years old is different in the type and degree of disease. Since wintering in the soil directly causes disease on the roots, it is difficult to control after planting ginseng, and the absenteeism rate damage due to disease is 21.8% for 4 years old and more than 50% for 6 years old, and 30-80% for 4 years old. It is evaluated as the largest enemy in the production of ginseng.

The initial symptom of ginseng root rot is that the leaf edges turn red or the leaves are constricted inward, and then die. Roots with such symptoms form a reddish brown or blackish brown lesion at the tip or middle, and the hairy roots turn reddish brown. Become corrupt. In addition, ginseng root rot is one of the biggest problems in domestic ginseng cultivation farms, as it occurs in all annuals from young ginseng to 6-year-olds.

Ginseng root rot, the main causative agent of ginseng root rot in Korea, is Cylindrocarpon destructans . Research is actively underway.

For the control of ginseng root rot, a method of using a soil fumigant and a control using chemical pesticides are known, but it is difficult to achieve a distinct effect with the above control method, and soil infectious pathogens such as ginseng root rot form thick film spores in the soil and are poor. Perfect control is very difficult because it can survive for a long time even under conditions. In addition, the fumigation method is not applicable when the root rot occurs due to root rot during ginseng cultivation, and there is a risk of destruction of the soil ecosystem by fundamentally removing harmful microorganisms and useful microorganisms. It is not easy to solve the root rot disease of ginseng because the control is low, and there is a possibility of seriously contaminating not only the cultivated ginseng but also the surrounding water and soil by indiscriminately abuse of chemical pesticides in some arable lands. Recently, the development of low-toxic, eco-friendly biopesticides has been attempted, but efforts for commercialization are required.

In addition, ginseng root rot disease is difficult to diagnose in the early stage due to slow translational progression, and even if the symptoms of plant wither appear, the disease is already prevalent in the roots and it is too late to control. Therefore, ginseng cultivation farmers prefer cropland and cropland with low possibility of disease in order to prevent damage from ginseng root rot. However, in major ginseng production areas, as grassland is gradually depleted due to an increase in cultivation area, the development of a means of discriminating grassland and cropland that has a low probability of ginseng root rot is increasingly required.

The existing identification of the presence of ginseng root rot bacteria in the soil is mainly performed in the form of posterior wall spores by inducing the growth of the pathogens present in the soil by using a selective medium, and then by a method of species identification through morphological characteristics and pathogenicity tests. I came, but it took a lot of time and manpower.

In addition, there was a problem that the germination rate of the posterior spores of Cylindrocarpon destructance in the soil was significantly low, making it difficult to detect pathogens or accurately measure the density, and the accuracy of determining whether or not infection was poor.

In particular, ginseng root rot germs occurring in domestic ginseng are also found in many hosts other than ginseng, and there are groups according to the pathogenic strength and weakness of ginseng. It takes a lot of time and manpower to predict the risk of occurrence. Species-specific with high detection sensitivity that can specifically amplify DNA for each strain of ginseng root rot according to pathogenic characteristics present in soil or plants for detection and species identification of pathogens that can solve the above problems. Development of markers and application techniques has been required.

Therefore, the inventors of the present invention are primers that can selectively bind to ginseng root rot germ DNA by effectively collecting pathogen DNA induced on soil and medium for accurate and rapid PCR species identification and detection of pathogens from soil, and ginseng using the primers. The present invention was completed by providing a method for detecting, counting, and diagnosing pathogenicity.

The problem to be solved by the present invention is to provide a method for more quickly and accurately detecting ginseng root rot bacteria (Cylindrocarpon destructans) and diagnosing pathogenicity.

In addition, another object of the present invention is to provide a primer set suitable for detecting and diagnosing pathogenicity of the ginseng root rot and a kit including the primer set.

In order to achieve the object of the present invention, the present invention is a primer set described in SEQ ID NO: 1 and SEQ ID NO: 2 and/or SEQ ID NO: 3 that can selectively bind only ginseng root rot bacteria according to the pathogenic properties present in the soil. And it provides a primer set for detecting and/or diagnosing pathogenicity of ginseng root rot selected from the group consisting of a primer set represented by SEQ ID NO: 4.

In addition, the present invention provides a kit for detecting and/or diagnosing pathogenicity of ginseng root rot including the primer set.

In addition, the present invention provides a method for detecting, counting and/or diagnosing pathogenicity of ginseng root rot using the primer set.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by comprising a primer set that reacts species-specifically with ginseng root rot and/or a primer set that reacts species-specifically to strong pathogenic pathogens. The primer set of the present invention is (1) 28S rDNA of ginseng root rot (Cylindrocarpon destructans ) among the IGS (intergenic spacer) and PC (pyruvate carboxylase) gene regions of 500 bp to 600 bp in Cylindrocarpon genus. And 18S rDNA of IGS (intergenic spacer) and PC (pyruvate carboxylase) genes are characterized by species-specific amplification.

The primer sets described in SEQ ID NO: 1 and SEQ ID NO: 2 can be used for detection of ginseng root rot, and the primer sets described in SEQ ID NO: 3 and SEQ ID NO: 4 are specifically bound to the ginseng root rot of strong pathogenic ginseng. It can function for diagnosis of pathogenicity of root rot.

The pathogenicity of the present invention was confirmed based on the morbidity rate and the seedling morbidity (depending on the morbidity, 1 to 5, 1: 20%, 2; 40%, 3: 60%, 4: 80%, 5: 100% morbidity) As a result, less than 5 is weakly pathogenic, and 5 can be isolated as a strong pathogenic strain.

Species CDPCF12 / CDPCR121 which specifically react with (SEQ ID NO: 1 / SEQ ID NO: 2) and CDIGS47NF2 / CDIGS47NR1 to respond only to one of the ginseng root rot fungal strain steel pathogenic strain (SEQ ginseng root rot fungus (Cylindrocarpon destructans) of the present invention No. 3/SEQ ID No. 4) In order to investigate the species specificity of ginseng root rot using a primer set, as publicly known strains, Alternaria genus, Aspergillus genus, Botrytis genus, Cladosporium genus, Coleto tree Kum, Didimella, Penicillium, Poma, Pyricularia, Pseum, Rhizoktonia, Screotinia, Screotium, Stemphylium, Trichoderma, Phytopsora, 27 species As a result of performing PCR on the strains, it was confirmed that species-specific amplification was performed with respect to the ginseng root rot strain (Figs. 2 to 5 and Table 4).

In addition, the CDPCF12/CDPCR121 (SEQ ID NO: 1/SEQ ID NO: 2) and CDIGS47NF2/CDIGS47NR1 (SEQ ID NO: 3/SEQ ID NO: 4) primer sets were used to perform specific amplification for ginseng root rot strains with different pathogenicity. It was confirmed that the amplification pattern was different depending on the pathogenicity (FIGS. 2 to 5 and Table 5).

The present invention provides a kit for detecting and diagnosing pathogenicity of ginseng root rot comprising a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2 and/or a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4.

The kit for detecting and diagnosing pathogenic ginseng root rot is a primer set (CDPCF12/CDPCR121) and/or SEQ ID NO: 3/ for species-specific amplification of the ginseng root rot (Cylindrocarpon destructans) strain. By including the primer set for diagnosing ginseng root rot of the primer set shown in SEQ ID NO: 4 (CDIGS47NF2/CDIGS47NR1), it is possible to amplify the DNA of the ginseng root rot of low density present in the soil quickly and accurately. It will enable diagnosis and establishment of effective control measures.

The present invention performs PCR (Polymerase Chain Reaction) on DNA isolated from a sample to detect ginseng root rot in the presence of a primer set for detecting and/or diagnosing pathogenicity of ginseng root rot ( Cylindrocarpon destructans); (2) It provides a method for diagnosing root rot of ginseng capable of detecting strong pathogenic pathogens, including the step of determining the presence or absence of root rot occurring in ginseng by analyzing the gene amplification by the PCR.

PCR used as a method of amplifying DNA may refer to a PCR that a person skilled in the art can utilize, such as conventional PCR, real-time PCR, and the primer set of the present invention. In an embodiment of the present invention, conventional PCR and real-time PCR are used. Was used.

In the present invention, the gene amplification by PCR is characterized in that the gene fragment is 80 to 150 bp in size, and the gene amplification is characterized by a primer set that reacts species-specifically with ginseng root rot.

The step of separating the DNA from the sample is characterized in that it is obtained from colonies generated after inoculation in a selection medium for ginseng root rot or directly isolated from crops or soil.

According to an embodiment of the present invention, in the step of separating the DNA, the colonies generated by selectively germinating on the medium using a selective medium for ginseng root rot from crops or soil are separated by NaOH DNA. It includes a technique for rapid and accurate detection and quantification using PCR by collecting DNA from spores in the soil of ginseng root rot by using PCR method after DNA collection and/or a soil DNA separation method applying MOBIO KIT. .

A method using a selective medium as a method for diagnosing the ginseng root rot according to an exemplary embodiment of the present invention is as follows. Ginseng root rot was collected and the soil was dried, 1 g of the soil was first mixed with 1 ml of sterilized water, and 100 μl was taken from the soil to make a second 1 ml of soil suspension. Subsequently, all of these secondary soil suspensions were plated and cultured on 3 PCNBA mediums each with 100 µl each. The resulting colonies are firstly observed to measure the density of bioactive spores in the soil by observing the colony's morphology, and after collecting the colony's DNA from the secondly generated colony using the NaOH DNA separation method, ginseng root rot using PCR It includes the step of confirming whether or not. According to another exemplary embodiment of the present invention, it includes a method of detecting and quantifying ginseng root rot bacteria by PCR by separating DNA by a method of adding a column procedure applying a method of separating MOBIO KIT from soil. The method using the selective medium enables the detection and approximate detection of bioactive spores, and the method using soil DNA separation enables the quantification of all spores present in the soil and can be quickly and accurately identified.

In addition, PCR was performed to confirm the detection sensitivity of ginseng root rot using the CDPCF12/CDPCR121 (SEQ ID NO: 1/SEQ ID NO: 2) and CDIGS47NF2/CDIGS47NR1 (SEQ ID NO: 3/SEQ ID NO: 4) primer sets. , The DNA amplification product to be amplified is a product having a size of 109 bp, and in the case of real-time PCR using SYBR green, it was confirmed that the set of CDPCF12/CDPCR121 (SEQ ID NO: 1/SEQ ID NO: 2) has a detection limit of approximately 1 pg ( 6, 7). In the case of CDIGS47NF2/CDIGS47NR1 (SEQ ID NO: 3/SEQ ID NO: 4), which generates a 117 bp amplification product, it was confirmed that it has a detection limit of approximately 10 fg (Figs. 8 and 9).

In addition, the present invention provides a first step of contacting a sample for counting ginseng root rot with a growth medium;

A second step of incubating the product of the first step; And

Including a third step of quantifying the ginseng root rot bacteria, the third step includes a method of counting ginseng root rot, characterized in that using the primer set of the present invention.

Detection and pathogenic diagnosis of ginseng root rot fungus comprising a primer set, and the primer set for diagnosing a ginseng root according to the present invention rot fungus (Cylindrocarpon destructans) detects, measures, and pathogenic diagnostic method, wherein the ginseng root rot bacteria detection and pathogenic The kit can reduce the time and cost required for diagnosing ginseng root rot, and increase the accuracy of the determination of infection, thereby reducing the risk and economic loss caused by root rot.

In addition to the newly developed method for separating a large amount of DNA in soil, conventional and real-time PCR detection techniques using primers specific to ginseng root rot species with high detection sensitivity are very useful for diagnosing and predicting the occurrence of ginseng root rot. It is believed to be useful.

Figure 1 shows the symptoms appearing in ginseng seedlings of 2-year-old roots by ginseng root rot when inoculated outdoors.
left ; Kang Pathogen (CY8001), right; Drug pathogenic bacteria (CY8014).
Figure 2 shows the conventional PCR species-specific reaction of the ginseng root rot ( Cylindrocarpon destructans ) species-specific detection primer CDPCF12 /CDPCR121. lane1: Cylindrocarpon destructans CY8001, lane 2-13: Alternaria, Aspergillus, Botrytis, Cladosporium, Coletotricum, Didimella, Penicillium, Forma genus, Pyricularia genus, Pseum genus, Rhizoktonia genus, Screotinia genus bacteria, NC: negative control.
3 shows the reaction of the primers CDPCF12/CDPCR121 on a ginseng root rot strain specific real-time PCR amplification curve.
4 shows conventional PCR specific reactions of primers CDIGS47NF2 / CDIGS47NR1 against strongly pathogenic ginseng root rot. Lane 1-8: Alternaria, Aspergillus, Botrytis, Cladosporium, Coletotricum, Didimella, Penicillium, Forma, lane 9: Drug pathogenic ginseng root rot Cylindrocarpon destructans CY8008, lane 10: Strong pathogenic ginseng root rot CY8001.
Figure 5 shows the reaction of the strong pathogenic ginseng root rot bacteria real-time PCR specific amplification curve of the primers CDIGS47NF2 / CDIGS47NR1.
6 shows the sensitivity of a real-time PCR reaction of CDPCF12/CDPCR121 to DNA of ginseng root rot. The genomic DNA of ginseng root rot was serially diluted in 7 steps of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg per ul, and then PCR was performed to show the reaction curve up to step 4.
7 shows a PCR standard curve using SYBR Green of CDPCF12/CDPCR121.
8 shows the sensitivity of a real-time PCR reaction of CDIGS47NF2/CDIGS47NR1 to DNA of ginseng root rot. The genomic DNA of ginseng root rot was serially diluted in 7 steps of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg per ul, and then PCR was performed to show the reaction sensitivity up to step 6 .
9 shows a PCR standard curve using SYBR Green of CDIGS47NF2/CDIGS47NR1.
10 shows the sensitivity of real-time PCR detection of CDIGS47NF2/CDIGS47NR1 against spores in the soil of ginseng root rot.
11 shows a real-time PCR standard curve for spores in the soil of ginseng root rot of CDIGS47NF2/CDIGS47NR1.
12 shows the sensitivity of real-time PCR detection of CDPCF12/CDPCR121 against spores in the soil of ginseng root rot.
13 shows a real-time PCR standard curve for spores in the soil of ginseng root rot of CDPCF12/CDPCR121.
14 shows the presence or absence of dependent identification and pathogenic strength using a primer set of CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1 and a real time PCR technique.

The present invention is a method for detecting, counting, and diagnosing pathogenicity of ginseng root rot (Cylindrocarpon destructans ), a primer set for detecting and/or diagnosing pathogenicity, and/or detection of ginseng root rot including the primer set. Alternatively, a kit for diagnosis of pathogenicity is provided.

Hereinafter, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the examples. It will be obvious to those skilled in the art that various modifications can be obtained with reference to the technical idea of the present invention and the following examples.

<Example 1> Strains and DNA preparation

1-1. Strain used

The DNAs of ginseng root rot (Cylindrocarpon destructans) and other strains used for the selection of species-specific primers were preserved in the Plant Pathology Laboratory of the Department of Agricultural Biology, Chungnam National University, and the publicly available strains sold to the Korea Agricultural Microbiological Resource Center were used. .

1-2. Pathogenicity investigation

Two-year-old seedlings were prepared to investigate the pathogenicity and differences of the strains isolated from diseased tissues and identified as ginseng root rot. As the inoculum, the strain was inoculated in PDA medium and cultured at 20°C for 14 days. Cultured mycelial tissue was ground with 40 ml of sterilized water to create a mycelial homogenous solution, and then 10 seedlings were immersed per each identified strain, and then transplanted into a package managed in a planned site located in Jinsan, Chungcheongnam-do. Pathogenicity was confirmed based on (denoted as 1 to 5 depending on the disease degree, 1: 20%, 2; 40%, 3: 60%, 4: 80%, 5: 100% morbidity) (FIG. 1). Lee Byung index = Lee Byung Joo X (Total of 10 seedlings / 10)/10. All strongly pathogenic strains completely destroyed the seedling roots in the soil. Pathogenicity by index was divided into strong pathogenicity: 5, severe pathogenicity: 3 or more and 5 or less, and weak pathogenicity: 3 or less.

1-3. DNA isolation

After pure separation of the specimens, in order to separate genomic DNA, potato dextrose broth (PDB) [potato 24g, dextrose 12g/1ℓ H2O] was inoculated in a medium and cultured at 20°C. The mycelium was harvested, divided into 1.5 ml e-tubes, lyophilized, ground, and then 400 µl of extraction buffer [200mM Tris-HCl (pH 8.0), 200mM NaCl, 30mM EDTA, 0.5% SDS] and Proteinase K (50µl) were added. And mixed well. Then, 2×CTAB solution [2% CTAB, 100mM Tris-HCl (pH 8.0), 20mM EDTA, 1.4M NaCl, 1% PVP] was added and mixed, and 600µl of chloroform was added, followed by 12,000rpm for 15 minutes. Centrifuged. The supernatant was separated by centrifugation and placed in a new tube, and 0.7 volume of isopropanol was added and left at room temperature for 10 minutes. This was centrifuged again at 12,000 rpm for 15 minutes to precipitate DNA, remove the remaining pellets, washed with 70% ethanol, and dried at room temperature until the ethanol was completely removed. 50 µl of sterile distilled water was added to the dried pellet and stored at -20°C until the next experiment. DNA separation for detecting pathogens from infected plants was performed according to the above procedure after dividing the plants into 1.5 ml tubes, lyophilizing them and grinding them.

1-4. Soil DNA isolation

Soil DNA separation can be performed using a known separation method and a commercially available kit. In this example, 0.2 g of rhizome soil within 10 cm of the paper part was added and 0.4 ml of sterilized water was added, and then impurities were dissolved at 40° C. for 10 minutes. Repeat centrifugation and removal 3 times, and then freeze-drying. Subsequently, DNA separation was performed according to the method of Mo-Bio (PowerSoil DNA isolation kit, USA), and finally, spin column treatment was added once and purified, and then stored at -20°C until the next experiment.

<Example 2> Design of primer for detecting ginseng root rot

For the selection of primers specific to the ginseng root rot fungus species from the PC gene (Pyrubate carboxylase) region, EMBL-EBI (http: //www.ebi.ac.uk) was analyzed using the clastralW program, and all ginseng root rot can be detected using the Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu). CDPCF12/CDPCR121 was designed as an appropriate primer set (Table 1). In addition, in order to select primers for detection of strong pathogenic strains, strains that showed strong pathogenicity and weak pathogenicity to ginseng that have already been pathogenically investigated were selected, and some bases in the IGS (rebosomal DNA intergenic spacer) region were compared with the same method as above. By analysis, primers CDIGS47NF2/CDIGS47NR1 specific only to strongly pathogenic strains within the ginseng root rot strain were designed (Table 1). All primers did not form hairpin loops inside the primers, did not form dimers or intenal loops between the primers, and were designed to have a size between 80-150bp, which can be used in both conventional PCR and real-time PCR.

Base sequence of the primer used in the present invention Sequence number Primer name DNA sequence (5'-3') One CDPCF12 CCC TGA ACG ATG TTG ACC AG 2 CDPCR121 CTT CTT GGG GTT GAG CAT GT 3 CDIGS47NF2 CGT CCT ACC CTA CAC CAC AAT 4 CDIGS47NR1 GCT TAG CCC ACT GTA AAT ACC

<Example 3> PCR detection and pathogenicity diagnosis using species-specific primers

All of the developed primer sets were subjected to PCR under the same conditions. After initial denaturation at 94°C for 5 minutes, the final 50 times were repeated with 1 cycle of 10 seconds at 94°C, 10 seconds at 60°C, and 30 seconds at 72°C, followed by 95°C to confirm the melting curve. After 10 seconds reaction at 0.5 ℃ was subjected to PCR reaction for 5 seconds to 65 ℃ intervals. The reaction composition conditions of conventional PCR (MJ Research Inc, PTC-100, USA) and real-time PCR (Bio-Rad, C1000, USA) are shown in Tables 2 and 3.

Conventional PCR reaction composition Component usage 10X Taq buffer 2 μl 10mM dNTP mix 0.4µl Forward primer (10pmol/µl) 1 μl Reverse primer (10pmol/µl) 1 μl Taq(5U/)(Toyobo, Blend Taq-plus, Japan) 0.1 μl Temp. DNA (2ng) 1 μl Total volume 20µl

Real-time PCR reaction composition Component usage SYBR premixture (Bio-Rad, iQ SYBR Green Supermix, USA) 10µl Forward primer (10pmol/µl) 1 μl Reverse primer (10pmol/µl) 1 μl Temp. DNA 1 μl Total volume 20µl

In order to investigate the species specificity of the developed primers, conventional and real-time PCR reactions were performed using CDPCF12/CDPCR121, CDIGS47NF2/CDIGS47NR1 primer sets for ginseng root rot strains and other plant pathogens isolated from ginseng root rot. .

Ginseng root rot fungus (Cylindrocarpon destructans) species specific detection primer CDPCF12 / CDPCR121 of conventional PCR species results of experiments for specific reaction, ginseng root rot fungus (Cylindrocarpon destructans) CY8001 (lane1) only specific to the reaction, Alter Canary subgenus , Aspergillus, Botrytis, Cladosporium, Coletotricum, Didimella, Penicillium, Poma, Pyricularia, Pseum, Rhyzoctonia, Screotinia It was found that it did not react to the bacteria (lanes 2 to 13). (FIG. 2)

Real-time PCR amplification results of primers CDPCF12/CDPCR121, Alternaria, Aspergillus, Botrytis, Cladosporium, Coletotricum, Didimella, Penicillium, Forma, Pyricle Although it does not react with bacteria of the genus Laria, Pseum, genus Rhizoctonia, and genus Screotinia, it can be seen that the amplification curve with a ct value of about 26 is shown for the tube in which the ginseng root rot germ DNA is present (Fig. 3). )

As a result of testing the conventional PCR specific reaction of the primers CDIGS47NF2 / CDIGS47NR1 against strongly pathogenic ginseng root rot bacteria, it reacts specifically only to strongly pathogenic ginseng root rot bacteria CY8001 (lane 10), and it reacts specifically to Alternaria genus, Aspergillus genus, Bo. Tritis, Cladosporium, Choletotricum, Didimella, Penicillium, and Forma (Lane 1-8) do not respond to, and show weak pathogenicity, ginseng root rot ( Cylindrocarpon destructans). ) It was confirmed that it did not react even to CY8008 (lane 9). (FIG. 4)

Real-time PCR amplification results of primers CDIGS47NF2 / CDIGS47NR1, Alternaria, Aspergillus, Botrytis, Cladosporium, Choletotricum, Didimella, Penicillium, Forma Although it does not react with pathogenic ginseng root rot (Cylindrocarpon destructans) CY8008 bacteria, for tubes with strong pathogenic ginseng root rot bacteria CY8001 DNA, an amplification curve with a ct value of about 20 could be confirmed (Fig. 5).

As a result of the experiment, it was confirmed that the primer sets of CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1 of the present invention were amplified species-specific with respect to the ginseng root rot strain (FIGS. 2 to 5).

In addition, PCR reaction specificity experiment using the primer set of ginseng root rot was confirmed for several strains, and the results are shown in Table 4.

PCR reaction specificity of primers CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1 against ginseng root rot Strain used CDPCF12
/CDPCR121 CDIGS47NF2
/CDIGS47NR1 Cylindrocarpon destructans CY8001 + + C. destructans CY8014 + - Fusarium culmorum - - F. equiseti - - F. eumartii - - F. graminearum - - F. lateritium - - F. moniliforme - - F. oxysporum f. sp. fragariae - - F. oxysporum f. sp. gladioli - - F. oxysporum f. sp. lycopersici - - F. oxysporum f. sp. niveun - - F. oxysporum f. sp. sesami - - F. proliferatum - - F. roseum - - F. semitectum - - F. solani from glycine - - F. solani from panax - - F. tricinctum - - F. udum - - F. anthophilum - - Alternaria alternata - - A. panax - - Armillaria calvescens - - Aspergillus niger - - Bipolaris oryzae - - Botrytis cinerea - - Cladosporium cucumerinum - - Colletotrichum acutatum - - Diaporthe ambigua - - Didymella bryoniae - - Glomerella cingulata - - Penicillium digitatum - - Phoma cucurbitacearum - - Phytophthora cactorum - - Pyricularia grisea - - Pythium ultimum - - Rhizoctonia solani (RG2) - - Rhizopus oryzae - - Scleotinia sclerotiorum - - Stemphylium versicarium - - Trichoderma hazianum - -

Using the CDPCF12/CDPCR121 primer set of the present invention, it was possible to perform rapid and accurate seed identification of ginseng root rot from DNA obtained from ginseng root rot disease (Figs. 2 to 5), and with a primer set of CDIGS47NF2/CDIGS47NR1. Is shown in Table 5 below for the pathogenic properties of strains that have been identified.

PCR reaction characteristics according to pathogenic characteristics of species-specific primer sets CDPCF12/CDPCR12 and CDIGS47NF2/CDIGSR1 against ginseng root rot Disclosure strain Lee Byung-soo PCR test result CDIGS47NF2/CDIGSR1 CDPCF12/CDPCR121 CY-8001 5.0 + + CY-8002 5.0 + + CY-8003 5.0 + + CY-8004 4.2 - + CY-8005 5.0 + + CY-8006 5.0 + + CY-8007 5.0 + + CY-8008 2.6 - + CY-8009 2.1 - + CY-8010 5.0 + + CY-8011 5.0 + + CY-8012 5.0 + + CY-8013 4.0 - + CY-8014 2.7 - + CY-8015 2.0 - + CY-8016 5.0 + + CY-8017 3.6 - + CY-8018 5.0 + + CY-8019 5.0 + + CY-8020 4.3 - + CY-8024 2.4 - + CY-8025 5.0 + + CY-8026 5.0 + + CY-8027 5.0 + + CY-8028 3.6 - + CY-8029 4.0 - + CY-8030 5.0 + + CY-8031 5.0 + + CY-8032 5.0 + + CY-8033 5.0 + + CY-8034 3.7 - + CY-8035 4.2 - + CY-8036 5.0 + + CY-8037 5.0 + + CY-8038 5.0 + + CY-8039 5.0 + + CY-8040 0.6 - +

+; positive, -; negative, 5 or less; Weak pathogenicity, 5; Kang pathogenicity.

<Example 4> Detection limit concentration of ginseng root rot sample

Using the primer sets of CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1, the minimum concentration capable of detecting ginseng root rot, that is, the sensitivity of the primer set was confirmed. To confirm the detection sensitivity of these species-specific primer sets, the genomic DNA of ginseng root rot was serially diluted in 7 steps per ul: 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg, and then each primer was Using the set, PCR was performed under the same conditions as the PCR reaction conditions of Example 3.

As a result, the detection sensitivity of the species-specific CDPCF12/CDPCR121 primer set against ginseng root rot in real-time PCR was Of the 7-step dilution samples of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg, the reaction occurred up to the DNA concentration, which is the fourth dilution step, indicating approximately 1 pg (Fig. 6). A PCR standard curve using SYBR Green of CDPCF12/CDPCR121 is shown in FIG. 7.

In addition, the detection sensitivity of the CDIGS47NF2/CDIGS47NR1 primer set, which can detect only strong pathogenicity among these pathogens, is up to 10fg, which is the 6th DNA dilution concentration among 7-step dilution samples of 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, and 1fg. It was confirmed that the amplification product appeared (FIG. 8), and a PCR standard curve using SYBR Green is shown in FIG. 9.

<Example 5> Quantification of sensitivity of detection of pathogens in soil using real-time PCR technique

To determine the minimum density of detectable ginseng root rot spores in the soil, dilute 10 times from the suspension of 10 ⁴ spores per gram of soil and mix to achieve a spore concentration of ^{10 4} , 10 ³ , 10 ² , 10 ¹ , 10 ^{0 contaminated soil.} After making, DNA was isolated from 200 mg of each soil, and real-time PCR was performed for the CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1 primer sets. As a result of real-time PCR, a reaction was shown to a soil sample mixed with 10 spores. The density of detectable lowest spores of CDIGS47NF2/CDIGS47NR1 was approximately 1-10 per gram (Fig. 10). A real-time PCR standard curve for spores in the soil of ginseng root rot of CDIGS47NF2/CDIGS47NR1 is shown in FIG. 11.

As a result of the experiment on the sensitivity of real-time PCR detection of CDPCF12/CDPCR121 against spores in the ginseng root rot germ soil, reactions were found to soil samples containing 100 spore concentrations. The density of detectable lowest spores of CDPCF12/CDPCR121 was approximately 100 per gram (Fig. 12). A real-time PCR standard curve for spores in the soil of ginseng root rot of CDPCF12/CDPCR121 is shown in FIG. 13.

<Example 6> PCR seeding identification and quantification technique of ginseng root rot present in soil

To determine the density of pathogens present in the diseased soil, the soil was dried and 1g was firstly mixed with 1 ml of sterilized water, and then 100µl was secondly taken to make 1ml of soil suspension. Then, 100 µl of each of these secondary soil suspensions were plated on 3 PCNBA media and cultured at 15°C (Table 6).

Selection medium for inducing the growth of ginseng root rot bacteria from diseased plants and soil Ingredients usage bacto peptone 5g agar 20g MgSO4 250mg KH2PO4 500mg PCNB 200mg penicillin G 50mg 85% lactic acid 1.3mL ethanol 20mL Na desoxycholate 130mg water 1L

DNA was separated according to the NaOH separation method of Table 7 from the colony induced on the medium, and was used for PCR seed identification.

Separation method using NaOH order Contents One Put 30mg-50mg of glass beads (0.25mm and 0.09-0.15mm 1:1 mix) in a 1.5ml tube. 2 Put 95µl of 0.5N NaOH into the tube, and add 30mg-50mg of the medium containing the hyphae. 3 Pellet pestle is used to sufficiently grind the medium containing mycelium. 4 Vortex vigorously for about 10 minutes using a finemixer. 5 Perform 13000rpm, 1min centrifuge. 6 Take 5µl of the supernatant and add 45µl of 100mM Tris-HCl (pH 8.0) to a new tube. 7 A total volume of 20 µl is used for 2µl PCR reaction solution.

The density of pathogens was confirmed by dividing the total number of pathogens appearing on all media of the secondary suspension identified using CDPCF12/CDPCR121 by the total media count and multiplying by 100 to estimate the total number of pathogens present in 1 g. DNA was obtained within 1 hour from the colonies formed on one of the three media, and the density of pathogens in the soil related to the presence or absence of species identification and pathogenic strength was estimated using primer sets of CDPCF12/CDPCR121 and CDIGS47NF2/CDIGS47NR1 (Fig. 14). ). In addition, as a result of PCR using CDPCF12/CDPCR121 for 6 colonies formed on one selective medium, colonies 1 and 2 exhibited positive reactions, so it was possible to identify them. As a result of performing a time PCR reaction, PCR-positive reaction was found only in colony 1, but it was found that strong pathogenic strains in the soil were also included. Therefore, through the PCR test using CDPCF12/CDPCR121, 8 ginseng root rot fungi could be identified from all the colonies derived from the secondary soil dilution in the same manner as above. In addition, it could be estimated that there were 2.33x100 live pathogen cells per gram of soil, and 25% of them were confirmed to be Kangwon by using the primer set of CDIGS47NF2/CDIGS47NR1, and both real-time and conventional PCR techniques could be used. Therefore, this technique is considered to be very useful in predicting the risk of disease occurrence of ginseng root rot caused by ginseng root rot, which is important in the selection of packaged sites.

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Claims (6)

delete delete Strong pathogenic ginseng root rot detection and pathogenic diagnostic kit comprising a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4.
A method for the detection and pathogenic diagnosis of strongly pathogenic ginseng root rot bacteria characterized by PCR amplification using primer sets represented by SEQ ID NO: 3 and SEQ ID NO: 4. delete delete

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