KR20110119038A - Species-specific primers to chinese cabbage clubroot pathogen spore and method for detecting or separating using thereof - Google Patents

Abstract

PURPOSE: A primer which specifically binds to clubroot and a method for diagnosing clubroot of Brassicaceae crops using the same are provided. CONSTITUTION: A method for diagnosing clubroot of Brassicaceae crops comprises: a step of performing PCR of DNA isolated from a spore of Brassicaceae crops from crops and soil under the presence of a primer set which specifically amplifies ITS(interual transcribed spacer) gene; and a step of analyzing gene amplification to determine club root of Brassicaceae crops. The primer set contains a forward primer of sequence number 1, 3, 5, or 7 and a reverse primer of sequence number 2, 4, 6, or 8.

Description

Specialty primers to chinese cabbage clubroot pathogen spore and method for detecting or separating using

The present invention relates to a primer specifically binding to root-knob spores, and a method for diagnosing root-knock disease of cruciferous crops using the same.

Chinese cabbage is one of the four major vegetable crops in Korea in terms of economy, and the domestic cultivation area is about 35,000 ha, accounting for 11.3% of the total vegetable cultivation area, and the output is about 2.3 million tons, the market size is about 1 It is estimated to reach trillion won.

Like this, Chinese cabbage, which is a very consumed and important crop, has been rapidly increasing incidence of root-knock disease (Wart disease) since the 1990s, and its host is not only cabbage, but also radish, cabbage, turnip, kale, gat, Broccoli, Arabidopsis, horseradish, beet, nasturtium and papaya, and the like, the root cause of the disease is also a infectious pathogen is known to survive for up to 15 years in the soil is difficult to diagnose and prevent early.

The morphological observation method using a fluorescence microscope has been used for the detection of root-knot bacteria, but it takes a lot of time to detect and requires expensive equipment. In order to solve the above problems, there is a need for a rapid and sensitive molecular biological detection method, which is recently used for detecting phytopathogens. However, specific primers were prepared by Ito et al. (2001) for the detection of root-knot bacteria, but their sensitivity was not applied to the soil.

Therefore, the present inventors endeavored to develop a method capable of quickly or accurately detecting or isolating root mycobacteria, and after selecting a primer that can selectively bind only root mycobacteria spores, using the primers to quickly and quickly The present invention was completed by discovering that it can be separated accurately.

It is an object of the present invention to provide a method for diagnosing root-knock disease of cruciferous crops, which can quickly and accurately separate root-mycosis spores by using a primer capable of selectively binding only root-knot spores.

SUMMARY OF THE INVENTION An object of the present invention is to provide a primer set for diagnosing root-knock disease of cruciferous crops, which can be used for diagnosing the root-knot disease of cruciferous crops, which can quickly and accurately isolate the root-knococcal spores.

In order to achieve the above object, the present invention provides a primer capable of selectively binding only spores of spores of pathogens and provides a method for diagnosing sprinkles of spp.

Hereinafter, a manufacturing method of the present invention will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples and may be exaggerated in order to sufficiently convey the spirit of the present invention to those skilled in the art.

Hereinafter, the technical and scientific terms used herein will be understood by those skilled in the art without departing from the scope of the present invention. Descriptions of known functions and configurations that may be unnecessarily blurred are omitted.

Hereinafter, the present invention will be described in detail.

The present invention

(1) Plasmodiophora brassicae ) crops or in the presence of a set of primers for diagnosing root-knock disease for species-specific amplification of an ITS (interactive transcribed spacer) gene present on at least one ribosome DNA operon selected from 18S rDNA, 5.8S rDNA and 28S rDNA of strains PCR (Polymerase Chain Reaction) is performed on DNA extracted from the spores of the root-knot disease strains taken from the soil;

(2) Analyzing the gene amplification by the PCR to determine the presence or absence of root-knock disease in cruciferous crops:

Provided is a method for diagnosing root-knock disease of cruciferous crops, comprising the steps.

The method for diagnosing the root-knock disease of the cruciferous crop of the present invention is an inter transcribed spacer (ITS) present on at least one ribosome DNA operon selected from 18S rDNA, 5.8S rDNA, and 28S rDNA of Plasmodiophora brassicae strain. Characterized by using a primer set for diagnosing root-knock disease to amplify the nucleotide sequence of the gene.

More specifically, the method for diagnosing the root-knock disease of the cruciferous crop of the present invention comprises at least one of SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6, and SEQ ID NO: 7 / SEQ ID NO: 8 Characterized by using a set of primers for the diagnosis of root-knock disease of the cruciferous crop containing.

The method for diagnosing the root nodule disease of the cruciferous crop of the present invention is a Plasmodiophora collected from a crop or soil. brassicae ) is characterized in that the isolates are selectively and quickly and accurately separated.

The present invention, in order to select a base sequence that can selectively bind to the root mycobacteria spores, Plasmodiophora ( Plasmodiophora) A primer was designed to amplify the nucleotide sequence of the ITS (interactive transcribed spacer) gene present on one or more ribosomal DNA operons selected from 18S rDNA, 5.8S rDNA and 28S rDNA of the brassicae ) strain. See FIGS. 1 and 2.

Plasmodiophora brassicae ) nucleotide sequence of the inter transcribed spacer (ITS) gene present on at least one ribosomal DNA operon selected from 18S rDNA, 5.8S rDNA and 28S rDNA of the strain is a species specific site that specifically binds to root spores spores , This is a method of detecting spores of root-mycosis spores has an important meaning in the present invention.

In the present invention, the primer set is Plasmodiophora Plasmodiophora brassicae ) from one or more forward primers selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 At least one reverse primer selected, and more particularly, the primer set is SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6 and SEQ ID NO: 7 / SEQ ID NO: 8 It characterized in that it comprises at least one of.

In the present invention, the method for diagnosing root-knock disease is Plasmodiophora ( Plasmodiophora) collected from crops or soil in a PCR reaction product. brassicae ) characterized in that it comprises DNA extracted from the spores of the strain.

More specifically, Alternaria sojae ), Fusarium oxysporum ), Didymella bryoniae ), Phytophthora infestans ), Pythium ultimum , Anthrax bacteria ( Colletotrichum) acutatum ), Bacterial fungus ( Botrytis cinerea ), Cylindrocarpon destructans ), Rhizoctonia solani ), Verticillium dahliae ) and Plasmodiophora Plasmodiophora using DNA extracted from spores of brassicae As a result of confirming the species specificity for brassicae ), Plasmodiophora Brassicae ) was not amplified at all except the infected plants, it was confirmed that specifically amplified only from the DNA of the root-knot bacteria. See FIG. 4.

In addition, in the present invention, the spores are characterized in that more than 10 ^-1 spore / μl concentration.

More specifically, after adjusting the spore suspension of Plasmodiophora brassicae to 10 ³ spores / μl, diluting the DNA was separated by 10-fold, and SEQ ID NO: 1 and SEQ ID NO: 2 (ITS1-1 / ITS1). -2) Real-time PCR was performed using primers. As a result, it was possible to detect up to 10 ⁻¹ , and it was confirmed that the detection sensitivity was significantly improved as well as the species specificity than the specific primer set developed by the conventional Faggian et al. (1999).

The present invention comprises at least one or more of SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6, and SEQ ID NO: 7 / SEQ ID NO: 8, a set of primers for the diagnosis of root-knock disease of cruciferous crops To provide.

The primer set is characterized by using the SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6 / SEQ ID NO: 7 / SEQ ID NO, which is specific for spore root fungal spores This is because only the spores of Plasmodiophora brassicae can be detected by including the nucleotide sequence that binds to each other.

Root-knock disease diagnosis method of cruciferous crops according to the present invention is very stable and easy to detect biological enzymes and physical elements, and the detection of root-knot bacteria spores in a short time can be quickly and accurately tested, Efficient control by the diagnosis of early root gall disease will be possible.

In addition, the primer set included in the method for diagnosing root-knock disease of cruciferous crops according to the present invention has species-specificity in the detection of root-knot bacteria spores, thereby greatly contributing to the molecular biologic study of root-knock disease. And prevention.

Figure 1 shows the information on the internucleotide sequence region of the ITS (root transcribed spacer) for the root-knot bacteria of the present invention,
Figure 2 shows a schematic diagram of the position of the primer for the diagnosis of root-knock disease of cruciferous crops according to the present invention,
Figure 3 shows the PCR amplification results for the root-knot bacteria using the root-knock disease diagnostic primer of the cruciferous crop according to the present invention,
Figure 4 shows the PCR amplification results of the species specificity for the root-knot bacteria using the root-knock disease diagnostic primer of the cruciferous crop according to the present invention,
(1: no treatment, 2: common soil infectious pathogens, 3: altenaria soybeans, 4: wilting germs, 5: vine blight germ, 6: potato blight germ, 7: rot fungus, 8: anthrax germ, 9 : Gray mold, 10: ginseng root rot, 11: tubercle rot, 12: root rot, 13: seed DNA)
Figure 5 shows the PCR amplification results for the detection folk degree according to the concentration of spore suspension using the root-knock disease diagnostic primer of the cruciferous crop of the present invention,
(1: 10 ⁴ , 2: 10 ³ , 3: 10 ² , 4: 10 ¹ , 5: 10 ^-1 , 6: 10 ^-2 , 7: 10 ^-3 . 8: 10 ^-4 )
6 is a result of confirming the species specificity of the root-knock disease diagnostic primer of the cruciferous crop of the present invention by real-time PCR,
(A: Species specificity analysis for root-knot bacteria, B: Melt peak value according to the results of A)
Figure 7 shows the results of real-time PCR amplification for the detection sensitivity according to the concentration of spore suspension using the root-knock disease diagnostic primer of the cruciferous crop of the present invention.
(A: analysis according to DNA concentration of Root-knot bacillus, B: standard curve value according to diluted DNA concentration of A)

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

[Example]

(1) Isolation of pure dormant spores

Plasmodiophora brassicae, used as a test strain, collected 500 root nodules from about 100 cabbage cultivation packages in 8 provinces except Jeju Island, and washed the root nodules several times with running water. The remaining soil particles and impurities were removed as much as possible.

The root nodules from which the impurities were removed were finely chopped using sterile scalpel and scissors, and then put into a thick test tube and finely crushed and homogenized by Homogenizer (IKA, 25 μm) while adding sterile water.

In Playa sumo video Fora beurasi roots organizations machul through the homogenization Kane (Plasmodiophora The dormant spores of brassicae strains were filtered through eight layers of gauze to separate dormant spore suspensions from the grounded plant tissue and soil residue together with cellulose nitrate to remove fine plant-derived impurities. filter method using a filter) and a syringe filter (Syringe filter) were used.

The separated dormant spore suspension was put into a syringe filter connected to a 8 μm cellulose filter and filtered by applying pressure through a vacuum pump, and the filtered suspension was put into a syringe filter connected to a 1 μm cellulose filter than dormant spores. Small microflora tissue and bacteria were separated.

The separated suspension was centrifuged at 3,500 rpm for 15 minutes to remove the supernatant, and then, sterile water was added to the obtained pellet and washed for 15 minutes in a centrifuge to obtain pure dormant spores in pellet form. , Stored at -20 ° C.

(2) Genomic DNA Isolation

The DNA was treated with 50 μl of DNase I (Sigma, D-4263) at a concentration of 50 units / ml, and then reacted at 37 ° C for 1 hour by applying 300 ppm of streptomycin and 100 ppm of rifampicin. Inactivation of DNase I was performed by treatment of 5 μl proteinase K (25 μg / μl) at 37 ° C. for 1 hour, followed by 300 μl of 5 mM EDTA and finally high temperature treatment at 80 ° C. hot block for 5 minutes. Finished. Spore suspension in which DNase I was inactivated was again centrifuged at 4,000 rpm for 15 minutes to remove supernatant and freeze-dried to use for DNA extraction.

After the pretreatment, the lyophilized pellets were ground with Pellet Pestile (Sigma) by adding glass beads (0.09 to 0.15 mm) to effectively break down the cell walls of fine dormant spores, and then extracting 400 [mu] l of buffer [200 mM Tris-Cl]. (pH 8.0), 200 mM EDTA, 1.4 M NaCl, 1% PVP] was added and extracted with 600 μl of chloroform. The extracted DNA was purified through a purification column (Intron Biotechnology, Korea) and dissolved in 100 μl of sterile water.

(3) species-specific primer  design

Plasmodiophora obtained from a region published in Genbank (http://www.ncbi.nlm.nih.gov) to design species-specific primers for improving detection sensitivity for dormant spores obtained from Example 1 above. Using the clathalW program in EMBL-EBI (http://www.ebi.ac.uk) using the sequence information of the ITS (inter transcribed spacer) region of Plasmodiophora brassicae , it was analyzed as shown in FIG. 1.

Species specific primers ITS1-1, ITS1-2 and ITS2-1 of Plasmodiophora brassicae using Oligonucleotide Properties Calculator (http://www.basic.northwestern.edu) , ITS2-2, PLF, PLR, PqF, PqR A total of four primer sets were designed as shown in Table 1 below. The synthesis of primers was commissioned by Genotech.

[Test Example 1] Examination of species specificity and sensitivity of primers by PCR amplification

(1) Species Specificity and Sensitivity of Primer by Conventional PCR Amplification

The primers of Table 1 were extracted from dormant spores isolated from three kinds of two cabbage root nodules and subjected to PCR amplification. For PCR reaction conditions, initial denaturation was performed at 94 ° C. for 5 minutes, followed by 35 cycles of denaturation 94 ° C./30 sec, annealing 65 ° C./20 sec, extension 72 ° C./20 sec, and final extension 72 ° C./5 min. .

As a result, as shown in the figure of Figure 3, it was confirmed that the primer set of Table 1 can all specifically amplify the DNA of Plasmodiophora brassicae ( Plasmodiophora brassicae ).

Specimen specificity was assayed using primer sets selected based on the results of FIG. 3,

The primer sets used were SEQ ID NO: 1 (ITS1-1) and SEQ ID NO: 2 (ITS1-2), which were present in the soil below to test for species specificity for Plasmodiophora brassicae . The main pathogens were investigated.

Alternaria devices sojae ), Fusarium oxysporum ), Didymella bryoniae ), Phytophthora infestans ), Pythium ultimum , Anthrax bacteria ( Colletotrichum acutatum ), Ash fungus ( Botrytis cinerea ), Ginseng root rot bacteria ( Cylindrocarpon destructans ), Rhizoctonia solanidah ( Rhizoctonia solanidah yl) ) And DNA were extracted from the spores of Plasmodiophora brassicae .

As a result, as can be seen in the figure of Figure 4, it was confirmed that only the DNA of the root-knot bacteria is amplified without any amplification except for the infected plants ( Plasmodiophora brassicae ).

From the above results, the primer of the present invention is also a result of confirming that species-specific detection of only Plasmodiophora brassicae .

In addition, SEQ ID NO: 1 (ITS1-1) and SEQ ID NO: 2 (ITS1-2) of the primer set of Table 1 were selected to assay for sensitivity in species-specific detection.

For detection sensitivity assay, dormant spores were adjusted to 10 ⁴ spores / μl using a hemocytometer, and then diluted DNA 10 times.

As a result, as can be seen from the results of Figure 5, it can be confirmed that up to 10 spores can be amplified, which significantly improves detection sensitivity as well as species specificity than the specific primer set developed by Faggian et al. (1999). I could confirm it.

(2) Examination of species specificity and sensitivity of primers by real-time PCR amplification

Based on the above PCR results, experiments were performed using real-time PCR to test more accurate species specificity and sensitivity of detection.

For real-time PCR, ITS1-1 and ITS1-2 primers were used. The total amount of PCR reaction solution was 20 μl, template DNA was used for 1 μl, primers of 2 were used, and iQTM SYBR Green Supermix from BIO-RAD was added. It was. PCR conditions were initial denaturation at 95 ° C for 5 minutes, 40 cycles of reaction at 95 ° C / 30 sec, 65 ° C / 20 sec, 72 ° C / 20 sec, and final extraction at 72 ° C for 5 sec. Calculated by increasing from 95 ° C to 95 ° C.

Six different soil pathogen DNAs and nine root-knot bacterium DNAs were tested for species specificity using species-specific primers ITS1-1 / ITS1-2, and as shown in FIG. The pathogen showed no PCR amplification curve, but only DNA of Plasmodiophora brassicae was amplified. In addition, all of the strains of Plasmodiophora brassicae ( Plasmodiophora brassicae ) all showed the same melt peak value of about 84 was confirmed that the PCR amplification was also stable.

In addition, after adjusting the spore suspension of Plasmodiophora brassicae to 10 ³ spores / μl in order to confirm the sensitivity of the detection, the DNA isolation was diluted 10-fold and the ITS1-1 / ITS1-2 primer. Real-time PCR was performed.

As a result, as can be seen in Figure 7, it was possible to detect up to 10 ^-1 , it was confirmed that the sensitivity has about 100 times higher than the conventional PCR of (4), the standard curve created by the cycle threshold value of the diluted DNA The value quantified the relative DNA concentrations across the samples, making the DNA concentration more intuitive.

From the above results, the species-specific primers of Table 1 of the present invention can not only detect species-specific Plasmodiophora brassicae but also effectively use them for molecular biological studies on root-knock disease with excellent detection sensitivity. Could confirm.

<110> Industry Foundation of Chungnam National University <120> Species-specific primers to chinese cabbage clubroot pathogen          spore and method for detecting or separating using understanding <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS1 forward primer <400> 1 ctagcgctgc atcccatatc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS1 reverse primer <400> 2 tgtttcggct aggatggttc 20 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ITS2 forward primer <400> 3 cggcatagct tgaacgaag 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ITS2 reverse primer <400> 4 gtgtgtgtcg atctgcgatt 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PLF <400> 5 tcctccgctt attgatatgc tt 22 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> PLR <400> 6 gaacctgcgg aaggatca 18 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PqF <400> 7 gcaagacaat gagctttgct g 21 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PqR <400> 8 tgtgtgtgtc gatctgcgat t 21

Claims (6)

(1) Species-specific amplification of the inter transcribed spacer (ITS) gene present on one or more ribosomal DNA operons selected from 18S rDNA, 5.8S rDNA and 28S rDNA of Plasmodiophora brassicae strain PCR (Polymerase Chain Reaction) is performed on the DNA extracted from the spores of the root-knot disease strain taken from the crop or soil in the presence of the primer set for root-knock disease diagnosis;
(2) Analyzing the gene amplification by the PCR to determine the presence or absence of root-knock disease in cruciferous crops:
A method of diagnosing root-knock disease of cruciferous crops, comprising the steps. The method of claim 1,
The primer set is Plasmodiophora brassicae ) from one or more forward primers selected from SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8 Root-knock disease diagnosis method of cruciferous crop, characterized in that it comprises one or more reverse primers selected. The method of claim 2,
The primer set includes at least one of SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6, and SEQ ID NO: 7 / SEQ ID NO: 8 How to diagnose the disease. The method according to any one of claims 1 to 3,
The method of diagnosing the root nodule disease is Plasmodiophora ( Plasmodiophora) collected from crops or soil in a PCR reaction product. brassicae ) A method for diagnosing root-knock disease of cruciferous crops, comprising DNA extracted from spores of a strain. The method of claim 4, wherein
The spore is a root-knock disease diagnosis method of cruciferous crop, characterized in that the concentration of more than 10 ^-1 spore / μl. A set of primers for the diagnosis of root-knock disease of cruciferous crops comprising at least one of SEQ ID NO: 1 / SEQ ID NO: 2, SEQ ID NO: 3 / SEQ ID NO: 4, SEQ ID NO: 5 / SEQ ID NO: 6, and SEQ ID NO: 7 / SEQ ID NO: 8.

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