KR101385106B1 - Disease diagnosis kit including species-specific primer, and detection and disease diagnosis method for Cylindrocarpon destructans - Google Patents

Abstract

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

Ginseng is botanically belonging to the genus Ginseng of araliace, and its scientific name is Panax ginseng C.A Meyer, a herbaceous plant whose roots are medicinal.

Korean ginseng (Panax ginseng C.A. Meyer) has been widely used in Korea, China, Japan and other countries for the purpose of health promotion. The effective components of ginseng are known to be significantly different depending on the type of ginseng, root area, harvest age, harvesting time, and manufacturing method. The ginseng root is generally known to have a high sugar content in low-grade roots and a high saponin content in high roots .

Ginseng is known to have fatigue recovery ability such as exercise ability, stress response effect, cognitive ability, psychological stability, etc. in Bahrke et al. (2000) ginseng research literature, it has a non-specific defense against foreign substances such as infectious substances in the body. It has been scientifically proven to have an immune enhancing effect as an activating substance.

Ginseng is a perennial semi-negative sage root (宿根 草), which is a semi-negative plant that requires only low-light light for photosynthetic photocompensation point of minimum 0.5klux. In addition, ginseng is a low-temperature plant that inhibits growth at high temperatures. Temperature is also an important growth limiting factor because it is weak to high temperature and high brightness, and the optimal growth temperature of Korean ginseng is estimated to be 18-21. Ginseng is evaluated as a plant that is difficult to cultivate due to its low disease resistance and low effective control methods in addition to the above-mentioned ingredients.

The plant of ginseng consists of roots and brains under the ground, stems, leaves, and flowers (fruits) on the ground. Since it is a perennial plant, sprouts emerge from the roots of the ground every spring, and the stems die every autumn. 착) Leave traces of being born every year. The growth and shape of the roots differ from year to year, and the harvest occurs at 4 to 6 years. Ginseng should be cultivated in the same field for 4-6 years for a long time, and after harvesting ginseng, it will not be grown any other crops, but after 1-2 years of cultivation planned site, it will be cultivated again and it will be damaged by various diseases. It is very large and the probability of serial failure is high.

Serial disturbance is Phytophthora cactorum ), ginseng root rot ( Cylindrocarpon) destructans ), Fusarium solani , Erwinia carotovora), Pseudomonas fluorine CL (Pseudomonas Pathogens such as fluorescens ) are known to occur because they act alone or in combination.

According to a recent study by the Institute of Agricultural Research, the ginseng root cause of ginseng more than 4 years old differs from the region and the type and extent of the disease, but the root rot disease is 35%, gray mold disease 32%, stem spot disease and 15%. Since it causes the disease directly on the roots as it overwinters in the soil, it is difficult to control after planting ginseng, and the damage rate from the disease is more than 21.8% for 4 years and 50% for 6 years, 30-80% for 4 years. It is evaluated as the biggest enemy in ginseng production.

The initial symptoms of ginseng root rot are reddening of leaf edges and repulsion of leaves, and these roots form reddish brown or blackish brown lesions at the ends or middle, and the hair roots turn reddish brown. Become corrupt. In addition, ginseng root rot is the biggest problem in domestic ginseng cultivation farms.

Ginseng root rot is a major cause of Korean ginseng root rot. Cylindrocarpon destructans ), which have been isolated from typical black lesions of serial disorders and have been confirmed pathogenicity, studies on the physiological characteristics and culture of bacteria have been actively conducted.

The use of soil fumigants and chemical pesticides for the control of ginseng root rot is known, but it is difficult to achieve a clear effect by the above control methods. Soil infectious pathogens such as ginseng root rot may form thick film spores in the soil. Perfect control is very difficult because it can survive long term. In addition, the fumigation method is not applicable when root rot occurs due to root rot during ginseng cultivation, and there is a risk of destruction of soil ecosystem by removing harmful microorganisms and useful microorganisms at the source. It is not easy to solve the root rot disease of ginseng because the control value is low and there is a possibility of seriously polluting not only the grown ginseng but also the surrounding water and soil by chemical pesticide indiscrete in some arable land. Recently, development of low-toxic, eco-friendly biopesticides has been attempted, but there is a need for commercialization.

In addition, the ginseng root rot disease is slow to develop before the early diagnosis, and the symptoms of plant wilting, even if the disease is already prevalent in the roots are often late to control. Therefore, ginseng cultivation farmers prefer cropland and low-probability crops to prevent disease caused by ginseng root rot. However, as the cropland is gradually depleted due to the increase in the cultivated area, the development of means for discriminating cropland and re-crop with low probability of ginseng root rot is increasingly required.

Determination of the presence of ginseng root rot bacteria in the soil is performed by the identification method through the morphological characteristics and pathogenicity test after inducing growth using selective medium for the pathogens present in the soil in the form of posterior spores. It took a lot of time and manpower for this.

In addition, the posterior spores of the Cyclodrocarpon dust lumps present in the soil had a low germination rate, making it difficult to detect pathogens or to accurately measure density, and thus, the accuracy of judging whether or not the infection was poor.

In particular, ginseng root rot bacteria that occur in domestic ginseng are found in many hosts other than ginseng, and there are groups according to the pathogenic strengths of ginseng. It takes a lot of time and manpower to forecast outbreak risks. Species-specific species with high detection sensitivity that can specifically amplify DNA for ginseng root rot bacteria strains according to pathogenic characteristics present in soil or plant for the detection and identification of pathogens that can solve the above problems The development of markers and utilization techniques has been called for.

Therefore, the present inventors effectively collect the pathogen DNA induced on the soil and the medium to identify and detect the PCR species of the pathogen from the soil effectively, and a primer capable of selectively binding to the ginseng root rot bacteria DNA and ginseng using the primer. The present invention has been completed by providing a method for diagnosing the root rot bacteria, counting and pathogenicity.

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

In addition, another object of the present invention is to provide a kit comprising a primer set and a primer set suitable for performing the detection and pathogenic diagnosis of the ginseng root rot bacteria.

In order to achieve the object of the present invention, the present invention is a primer set and / or SEQ ID NO: 3 described in SEQ ID NO: 1 and SEQ ID NO: 2 which can selectively bind only ginseng root rot bacteria according to the pathogenic properties present in the soil And a set of primers for detecting and / or pathogenic diagnosis of ginseng root rot bacteria selected from the group consisting of primer sets set forth in SEQ ID NO: 4.

In another aspect, the present invention provides a kit for the detection and / or pathogenic diagnosis of ginseng root rot bacteria comprising the primer set.

The present invention also provides a method for detecting, counting and / or pathogenicity of ginseng root rot bacteria using the primer set.

Hereinafter, the present invention will be described in detail.

The present invention is characterized in that it comprises a primer set for species-specific reaction with ginseng root rot bacteria and / or a primer set for species-specific reaction to strongly pathogenic pathogens. Pre-set timer of the present invention (1) cylinder throw car phone in 500 bp to 600 bp in size IGS (intergenic spacer), PC (pyruvate carboxylase) gene from ginseng root rot fungi in the area (Cylindrocarpon) (Cylindrocarpon destructans ) of 28S rDNA, 18S rDNA IGS (intergenic spacer), PC (pyruvate carboxylase) genes are characterized in that the species-specific amplification.

The primer sets described in SEQ ID NO: 1 and SEQ ID NO: 2 can be utilized for the detection of ginseng root rot bacteria, and the primer sets described in SEQ ID NO: 3 and SEQ ID NO: 4 ginseng by specifically binding to the pathogenic ginseng root rot bacteria Can function as a pathogenic diagnosis of root rot bacteria.

Pathogenicity of the present invention is confirmed based on the morbidity rate and degree of seedlings (1 to 5 depending on the degree of disease, 1: 20%, 2; 40%, 3: 60%, 4: 80%, 5: 100% disease) Less than 5 degrees of pathogenicity, weakly pathogenic, 5 may be separated into a strongly pathogenic strain.

Ginseng root rot bacteria of the present invention ( Cylindrocarpon) destructans ) CDPCF12 / CDPCR121 (SEQ ID NO: 1 / SEQ ID NO: 2) and CDIGS47NF2 / CDIGS47NR1 (SEQ ID NO: 3 / SEQ ID NO: 4) primer sets that only react to highly pathogenic strains of ginseng root rot In order to investigate species specificity against ginseng root rot by using the strains, the genus Alternaria, Aspergillus, Botrytis, Cladosporium, Choletotricum, Didimela, Penicillium PCR was performed on 27 strains of the genus, Forma, Pycuricaria, Psium, Laytononia, Screotinia, Screotium, Stemphyllium, Trichoderma and Pytopsora. It was confirmed that the species-specific amplification for the ginseng root rot bacteria strain (Fig. 2 to 5 and Table 4).

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 strains of ginseng root rot bacteria having different pathogenicities. It was confirmed that the amplification pattern is different depending on the pathogenicity (FIGS. 2 to 5 and Table 5).

The present invention provides a kit for detecting and pathogenic ginseng root rot comprising a primer set described in SEQ ID NO: 1 and SEQ ID NO: 2 and / or a primer set described in SEQ ID NO: 3 and SEQ ID NO: 4.

The ginseng root rot detection and pathogenic diagnostic kit includes a primer set (CDPCF12 / CDPCR121) set forth in SEQ ID NO: 1 / SEQ ID NO: 2 that species-specific amplifies the strain of Cylindrocarpon destructans and / or SEQ ID NO: 3 / By including the primer set for diagnosing ginseng root rot disease of the primer set (CDIGS47NF2 / CDIGS47NR1) set forth in SEQ ID NO: 4, the DNA of the low density ginseng root rot bacterium present in the soil can be amplified quickly and accurately. It will enable the diagnosis and establishment of effective control measures.

The present invention ginseng root rot bacteria ( Cylindrocarpon) depolymerans ) PCR (Polymerase Chain Reaction) is performed on DNA isolated from a sample to detect ginseng root rot bacteria in the presence of a primer set for detecting and / or pathogenic diagnostics; (2) analyzing the gene amplification by PCR and determining the presence or absence of root rot disease occurring in ginseng, and provides a method for diagnosing root rot disease of ginseng that can detect strongly pathogenic pathogens.

 PCR used as a method for amplifying DNA may mean a PCR that can be used by those skilled in the art, such as conventional PCR, real-time PCR, primers, in one embodiment of the present invention, conventional PCR, Real-time PCR Used.

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

Separating the DNA from the sample is characterized in that it is obtained directly from the crop or soil, or obtained from colonies generated after inoculation in ginseng root rot selective medium.

According to an embodiment of the present invention, the step of separating the DNA from the crop or soil by using the ginseng root rot selective medium, the spores of the biomass present in the soil selectively germinated on the medium produced NaOH DNA separation method The method for detecting DNA by using PCR method after collecting DNA and / or the soil DNA separation method using MOBIO KIT, and the method for rapid and accurate detection and quantification using PCR by collecting DNA of spores in soil of ginseng root rot bacteria .

Method of using a selection medium as a method for diagnosing the ginseng root rot bacteria according to an exemplary embodiment of the present invention is as follows. Root-root soil of the area suspected of ginseng root rot was collected, dried, and 1 g of the soil was mixed with 1 ml of sterile water, and 100 µl was taken from the 1 ml soil suspension. Subsequently, all of these secondary soil suspensions were plated in three PCNBA medium at 100 μl and cultured. The produced colonies were first observed by morphology of the spores in the soil, and then the DNA of the colonies was collected by NaOH DNA separation from the secondly produced colonies. Checking the availability of the. According to another exemplary embodiment of the present invention, a method of detecting and quantifying ginseng root rot bacteria by PCR is performed by separating DNA by a column method applying a method of separating MOBIO KIT from soil. The method using selective medium can detect and approximate bioactive spores, and the method using soil DNA separation can quantify the whole spores present in the soil and can be confirmed quickly and accurately.

In addition, PCR was performed to confirm the detection sensitivity against ginseng root rot bacteria 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 was a product of 109 bp, and in case of real-time PCR using SYBR green, the CDPCF12 / CDPCR121 (SEQ ID NO: 1 / SEQ ID NO: 2) set had a detection limit of approximately 1 pg ( 6, 7). In the case of CDIGS47NF2 / CDIGS47NR1 (SEQ ID NO: 3 / SEQ ID NO: 4) generating an amplification product of 117 bp, it was confirmed that the detection limit was approximately 10 fg (FIGS. 8 and 9).

In another aspect, the present invention is the first step of contacting the sample to be counted ginseng root rot bacteria with the growth medium;

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

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

Cylindrocarpon destructans detection, counting and pathogenicity diagnostic method according to the present invention, ginseng root rot bacterium detection and pathogenicity diagnostics and primer set for diagnosing and pathogenicity detection and pathogenic diagnosis comprising the primer set The kit can reduce the time and cost required to diagnose ginseng root rot bacteria and improve the accuracy of infection determination, thereby reducing the risk and economic loss caused by the root rot disease.

In the present invention, conventional and real-time PCR detection techniques using primers specific to ginseng root rot bacteria with high detection sensitivity as well as a large amount of DNA separation method in the soil are very useful for the diagnosis and prediction of disease occurrence in ginseng root rot. It is expected to be useful.

Figure 1 shows the symptoms appearing in two-year-old ginseng seedlings by ginseng root rot bacteria at outdoor inoculation.
left ; Strong pathogenic bacteria (CY8001), right; About pathogenic bacteria (CY8014).
2 is a ginseng root rot bacteria ( Cylindrocarpon) destructans ) shows a conventional PCR species-specific reaction of the species-specific detection primer CDPCF12 / CDPCR121. lane1: Ginseng root rot fungus (Cylindrocarpon destructans ) CY8001, lane 2-13: Alternaria, Aspergillus, Botrytis, Cladosporium, Colletotricum, Didimela, Penicillium, Poma, Pycuricaria, Pseumium, Laytononia, Screotinia, NC: negative control.
Figure 3 shows the ginseng root rot bacteria species specific real-time PCR amplification response of the primer CDPCF12 / CDPCR121.
Figure 4 shows the conventional PCR specific response to the strongly pathogenic ginseng root rot bacteria of the primer CDIGS47NF2 / CDIGS47NR1. Lane 1-8: Alternaria, Aspergillus, Botrytis, Cladosporium, Colletotricum, Didimela, Penicillium, Forma bacteria, lane 9: About pathogenic ginseng root rot Cylindrocarpon destructans CY8008, lane 10: Pathogenic ginseng root rot bacteria CY8001.
Figure 5 shows the strongly pathogenic ginseng root rot bacteria real-time PCR specific amplification curve response of the primer CDIGS47NF2 / CDIGS47NR1.
Figure 6 shows the sensitivity of real-time PCR reaction to the ginseng root rot bacteria DNA of CDPCF12 / CDPCR121. The genomic DNA of ginseng root rot bacteria was serially diluted in 7 steps of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg per ul, followed by PCR.
7 shows a PCR standard curve using SYBR Green of CDPCF12 / CDPCR121.
Figure 8 shows the sensitivity of real-time PCR reaction to the ginseng root rot bacteria DNA of CDIGS47NF2 / CDIGS47NR1. The genomic DNA of ginseng root rot bacteria was serially diluted in 7 steps of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per ul, followed by PCR .
9 shows a PCR standard curve using SYBR Green of CDIGS47NF2 / CDIGS47NR1.
Figure 10 shows the sensitivity of real-time PCR detection for spores in the ginseng root rot bacteria soil of CDIGS47NF2 / CDIGS47NR1.
Figure 11 shows the real-time PCR standard curve for spores in the ginseng root rot disease soil of CDIGS47NF2 / CDIGS47NR1.
Figure 12 shows the sensitivity of real-time PCR detection of spores in the ginseng root rot pathogen soil of CDPCF12 / CDPCR121.
Figure 13 shows the real-time PCR standard curve for spores in the ginseng root rot bacteria soil of CDPCF12 / CDPCR121.
FIG. 14 shows the identification of pathogens and pathogenic agents using CDPCF12 / CDPCR121 and CDIGS47NF2 / CDIGS47NR1 primer sets and real time PCR.

The present invention ginseng root rot bacteria ( Cylindrocarpon) destructans ) detection, counting and pathogenic diagnostic methods, a primer set for detecting and / or diagnosing ginseng root rot bacteria and a kit for detecting and / or pathogenic diagnostics of ginseng root rot bacteria comprising the primer set.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples. It will be apparent to those skilled in the art that various modifications can be made with reference to the technical spirit of the present invention and the following examples.

Example 1 Strain and DNA Preparation

1-1. Used strain

The DNAs of Cylindrocarpon destructans and other strains used for the selection of species-specific primers were used as strains preserved in the Department of Agricultural Biology and Plant Pathology, Chungnam National University, and the public strains distributed by the Korea Agricultural Microbiological Resource Center. .

1-2. Pathogenicity investigation

Two-year-old seedlings were prepared to investigate the pathogenicity and differences of the strains identified as ginseng root rot by separating them from diseased tissues. The inoculum was used after inoculating the strain on PDA medium for 14 days at 20 ° C. The cultured mycelial tissue was pulverized with 40 ml of sterile water to make a mycelial homogenate. After dipping 10 seedlings per individual strain, they were transplanted into a managed package at a planned site in Jinsan, Chungnam. Pathogenicity was confirmed based on (labeled 1 to 5 according to the degree of disease, 1: 20%, 2; 40%, 3: 60%, 4: 80%, 5: 100% disease) (FIG. 1). Byeongjeom Index = Byeongju Lee X (10 total seedlings and Byeong / 10) / 10. All strongly pathogenic strains completely destroyed seedling roots in the soil. Pathogenicity was classified into strong pathogenicity: 5, moderate pathogenicity: more than 3 and less than 5, and weak pathogenicity: less than 3.

1-3. DNA  detach

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

1-4. Soil DNA Isolation

Soil DNA separation may use a known separation method and a commercially available kit. In this example, 0.2 g of root zone soil in 10 cm of paper is added and 0.4 ml of sterile water is added, followed by dissolving impurities at 40 ° C. for 10 minutes. Centrifugation is repeated three times, followed by lyophilization. Subsequently, DNA isolation was performed according to the method of Mo-Bio (PowerSoil DNA isolation kit, USA), and finally, one spin column treatment was added and purified, and then stored at -20 ° C until the next experiment.

Example 2 Design of a primer for detecting ginseng root rot bacteria

EMBL-EBI (http: http://www.embl-ebi.com/) for DNA sequences of the same gene region of cylindrocaphone albumin and cylindrocapon hateronema strains for selection of ginseng root rot species specific primers from the Pyrubate carboxylase region. Species specific for all ginseng root rots can be detected using the clastralW program at //www.ebi.ac.uk using the Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu). CDPCF12 / CDPCR121 was designed with the appropriate primer set (Table 1). In addition, to select primers for the detection of strongly pathogenic strains, strains that showed strong pathogenicity and weak pathogenicity against ginseng that had already been examined for pathogenicity were selected, and some bases of IGS (intergenic spacer) regions were compared in the same manner as above. The primers CDIGS47NF2 / CDIGS47NR1 specific to the highly pathogenic strains of ginseng root rot bacteria were analyzed (Table 1). The primers were designed to be between 80-150bp in size without the formation of hairpin loops inside the primers, and no dimer or intenal loops were formed between the primers.

Used in the present invention Primer  Base sequence SEQ ID NO: Name of the primer 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> Species Specific Primer  Used PCR  Detection and Pathogenic Diagnosis

All primer sets developed were subjected to PCR under the same conditions. After initial denaturation at 94 ° C. for 5 minutes, 10 cycles at 94 ° C., 10 seconds at 60 ° C., and 30 seconds at 72 ° C. were repeated for the last 50 times, followed by 95 ° C. to confirm the melting curve. After 10 seconds in the reaction was PCR reaction for 5 seconds to 65 ℃ at 0.5 ℃ interval. 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 0.4 μl Forward primer (10 pmol / μl) 1 μl Reverse primer (10 pmol / μl) 1 μl Taq (5U /) (Toyobo, Blend Taq-plus, Japan) 0.1 μl Temp. DNA (2ng) 1 μl Total volume 20 쨉 l

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

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

Ginseng Root Disease destructans ) Species-specific detection primers CDPCF12 / CDPCR121 were tested for conventional PCR species-specific reactions. Cylindrocarpon destructans ) Reacts specifically to CY8001 (lane1), Alternaria, Aspergillus, Botrytis, Cladosporium, Colletotricum, Didimela, Penicillium, Poma, Flute It was found that it did not react to the genus Cularia, Psium, Lysatonia, Screotinia (lanes 2 to 13) (FIG. 2).

Real-time PCR amplification of CDPCF12 / CDPCR121 primers, Alternaria, Aspergillus, Botrytis, Cladosporium, Choletotricum, Didimela, Penicillium, Poma, Pyricul Although it does not react with the bacteria of the genus Laria, Psium, Lysatonia, and Screotinia, it can be seen that the tube with the ginseng root rot bacterium DNA shows an amplification curve with a ct value of about 26 (FIG. 3). )

As a result of the conventional PCR-specific reaction of the highly pathogenic ginseng root rot bacteria of the primer CDIGS47NF2 / CDIGS47NR1, it reacted only specifically to the pathogenic ginseng root rot bacteria CY8001 (lane 10), genus Alternaria, Aspergillus, Bo Ginseng Root Bacterium ( Cylindrocarpon ) that does not respond to Tritis, Cladosporium, Colletotricum, Didimela, Penicillium, and Forma (Lane 1-8) and also exhibits pathogenicity destructans ) did not react to CY8008 (lane 9) (Fig. 4).

Real-time PCR amplification of the primer CDIGS47NF2 / CDIGS47NR1, genus Alternaria, Aspergillus, Botrytis, Cladosporium, Choletotricum, Didimela, Penicillium, Poma Amplification curves with a ct value of about 20 were observed for the tube in which the pathogenic Ginseng Root Bacterium CY8008 does not react with the pathogenic Ginseng Root Bacterium CY8008.

The primer set of the CDPCF12 / CDPCR121, CDIGS47NF2 / CDDIGS47NR1 of the present invention was confirmed that the species-specific amplification for the ginseng root rot bacteria strains (Fig. 2 to 5).

In addition, PCR reaction specificity experiments using the primer set of ginseng root rot bacteria were confirmed for various strains, and the results are shown in Table 4.

PCR reaction specificity of ginseng root rot bacteria of primers CDPCF12 / CDPCR121 and CDIGS47NF2 / CDIGS47NR1 Used strain 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 - - Sccleotinia sclerotiorum - - Stemphylium versicarium - - Trichoderma hazianum - -

Using the CDPCF12 / CDPCR121 primer set of the present invention, rapid and accurate identification of ginseng root rot bacteria could be performed from DNA obtained from the ginseng root rot disease plant (FIGS. 2 to 5), and the primer set of CDIGS47NF2 / CDIGS47NR1 Table 3 shows the pathogenic characteristics of the strains that have been identified as follows.

PCR Reaction Characteristics of Species-Specific Primer Set CDPCF12 / CDPCR12 and CDIGS47NF2 / CDIGSR1 Against Ginseng Root Disease Published strain Byeong index 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; Drug pathogenic, 5; Strong pathogenicity.

< Example  4> Ginseng root rot  Detection limit concentration of the sample

The primer sets of CDPCF12 / CDPCR121 and CDIGS47NF2 / CDIGS47NR1 were used to confirm the minimum concentration, that is, the sensitivity of the primer set, to detect ginseng root rot bacteria. To confirm the detection sensitivity of these species-specific primer sets, the genomic DNA of ginseng root rot bacteria was serially diluted in seven steps of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, 1 fg per ul, and then each primer was PCR was performed under the same conditions as the PCR reaction conditions of Example 3 using the set.

As a result, the detection sensitivity of the species-specific CDPCF12 / CDPCR121 primer set against ginseng root rot bacteria in real-time PCR was The reaction showed up to the DNA concentration, which is the fourth dilution step, among the seven dilution samples of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg, indicating that the reaction was approximately 1 pg (FIG. 6). The 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 approximately 6 th DNA dilution concentration of 10 fg in 7 diluting samples of 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg. It was confirmed that the amplification product appeared (FIG. 8), and the PCR standard curve using SYBR Green is shown in FIG. 9.

< Example  5> Real - time PCR  Technique In soil  Pathogen Detection Sensitivity Quantification

To determine the minimum density of detectable ginseng root rot spores in soil, dilute 10 times from 10 ⁴ spore suspensions per gram of soil and mix them to a concentration of 10 ⁴ , 10 ³ , 10 ² , 10 ¹ , 10 ⁰ After the DNA was separated from each soil 200mg, real-time PCR was performed on CDPCF12 / CDPCR121 and CDIGS47NF2 / CDIGS47NR1 primer sets. As a result of real-time PCR, it showed reaction to soil samples containing 10 spores. The density of detectable trough spores of CDIGS47NF2 / CDIGS47NR1 was approximately 1-10 per g (FIG. 10). The real-time PCR standard curve for spores in the ginseng root rot bacteria of CDIGS47NF2 / CDIGS47NR1 is shown in FIG. 11.

As a result of the real-time PCR detection sensitivity of CDPCF12 / CDPCR121 to the spores in the ginseng root rot bacteria, soil samples containing 100 spore concentrations appeared. The density of detectable trough spores of CDPCF12 / CDPCR121 was about 100 per g (FIG. 12). The real-time PCR standard curve for spores in ginseng root rot bacteria of CDPCF12 / CDPCR121 was shown in FIG. 13.

< Example  6> present in soil Ginseng root rot PCR Follower  And quantification techniques

To determine the density of pathogens present in the diseased soil, 1g of the soil was dried and mixed in 1ml of sterile water. Then, 100µl was taken from the 2nd to make 1ml of soil suspension. These secondary soil suspensions were then plated in three PCNBA medium at 100 μl each and incubated at 15 ° C. (Table 6).

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

DNA was isolated from the bacterium induced on the medium according to the NaOH separation method of Table 7 and used for PCR identification.

Separation Method Using NaOH order Contents One Add 30mg-50mg (0.25mm and 0.09-0.15mm 1: 1 mix) glass beads to the 1.5ml tube. 2 Add 95µl of 0.5N NaOH to the tube and add 30mg-50mg of medium fragment containing mycelium. 3 Pellet pestle is used to grind enough pieces of media containing mycelia. 4 Vortex strongly for 10 minutes using Finemixer. 5 Perform 13000 rpm, 1 min centrifuge. 6 Take 5 µl of the supernatant and add 45 µl of 100 mM Tris-HCl (pH 8.0) to a new tube. 7 2 μl of total volume 20 μl PCR reaction solution is used.

CDPCF12 / CDPCR121 was used to determine the density of pathogens by estimating the total number of pathogens present in 1 g by dividing the total number of pathogens on all media of the secondary suspension identified by the total medium index and multiplying by 100. DNA was obtained within 1 hour from the bacterial flora formed on one of the three media, and the density of pathogens in soil associated with the identification and pathogenic potents was estimated using primer sets of CDPCF12 / CDPCR121 and CDIGS47NF2 / CDIGS47NR1 (FIG. 14). ). In addition, PCR was carried out using CDPCF12 / CDPCR121 on 6 bacterial colonies formed on one selective medium. As a result, the colonies 1 and 2 showed positive reactions, and these were again identified using primer sets of CDIGS47NF2 / CDIGS47NR1. As a result of the time PCR reaction, PCR positive reaction was observed only in the first flora, but it was also found that strongly pathogenic strains were included in the soil. Therefore, 8 ginseng root rot bacteria could be identified by PCR test using CDPCF12 / CDPCR121 from all the bacterial flora derived from the three soil dilutions from the second soil dilution. In addition, it was estimated that there were 2.33x100 live pathogen cell counts per gram of soil, and 25% of them were identified as strong hospitals using the CDIGS47NF2 / CDIGS47NR1 primer set, and both real-time and conventional PCR techniques could be utilized. Therefore, this technique is very useful for predicting the risk of ginseng root rot caused by ginseng root rot.

Attach an electronic file to a sequence list

Claims (9)

delete delete delete delete delete delete Separating DNA from a sample to detect ginseng root rot bacteria;
Screening ginseng root rot bacteria by performing PCR (Polymerase Chain Reaction) using a primer set represented by SEQ ID NO: 1 and SEQ ID NO: 2; And
Analyzing the amplified gene by performing PCR using a primer set represented by SEQ ID NO: 3 and SEQ ID NO: 4 for the selected ginseng root rot bacteria
Method of detecting ginseng root rot bacteria against strong pathogenicity. delete delete

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