KR20020004659A - A Molecular Marker for Colletotrichum gloeosporioides in Sweet persimmon - Google Patents

Abstract

PURPOSE: A molecular marker, particularly, DNA marker containing a pair primer, ITS-4 and CO-1, is provided which is used for the diagnosis of anthracnose disease in a sweet persimmon. It detects the infection of anthrax bacteria, Colletotrichum spp, at a minimum concentration of 10pg, thereby diagnosing anthracnose disease at its early stage and consequently preventing over-use of pesticides for anthracnose control. CONSTITUTION: The anthrax bacterium, Colletotrichum gloeosporioides, causes anthracnose disease in a sweet persimmon. The DNA marker for the diagnosis of anthracnose disease in a sweet persimmon is produced by amplification using a pair primer ITS-4 having nucleotide sequence of SEQ ID NO:1 (tcctccgctt attgatatgc), and CO-1 having nucleotide sequence of SEQ ID NO:2 (ataaccttt gtgaacatac).

Description

Molecular Marker for Diagnosing Persimmon Anthrax {A Molecular Marker for Colletotrichum gloeosporioides in Sweet persimmon}

The present invention relates to a molecular marker for diagnosing persimmon anthrax, and more particularly, to a molecular marker for diagnosing persimmon anthrax, including ITS-4 and CO-1, which are highly conserved with primer pairs.

As of 1998, domestic sweet persimmons have a cultivated area of 23,000 ha and output of 208,900 M / T, which occupies the main positions of domestic fruits. Persimmon tree caused by the genus Collettricum (Colletotrichum.spp), which has never been a problem in Gyeongsangnam-do, North-North, and Jeolla-do since 1990 due to the continuous increase in the area of persimmon tree and the bias of single varieties. Anthrax is a frequent occurrence, and the pathogen has a wide host range including persimmon trees, which causes serious detrimental effects on fruit growth and flowering in the following year. Persimmon anthracnose pathogens, along with circular pattern deciduous disease, are the main diseases of persimmon trees.

However, since the prior art relies only on microscopic or visual observation, there has been a problem that can be detected only when the disease has progressed to some extent. As a result, current farms use excessive pesticides to prevent anthrax, causing problems such as water pollution as well as pollution of the soil environment due to soil residues of pesticides. Therefore, in the conventional method, it is difficult to detect anthrax pathogens early because the anthracnose pathogens germinate on persimmon leaves or fruits and the anthrax pathogens are examined through microscopic observation or visual observation only after the disease has been advanced to some extent. As a result, there is a problem that can miss the timing of control when considering the rate of disease progression, there is a need for improvement.

Therefore, the inventors of the present invention have made a thorough research to solve the above problems, as a result of amplifying by using a highly conserved ITS and CO-1 region as a primer (primer) as a site of severe mutation in fungi The present invention has been completed by confirming that it is possible to design diagnostic molecular markers capable of specifically detecting pathogens.

After all, the main object of the present invention is to provide a molecular marker for diagnosing persimmon anthrax using ITS-4 and CO-1 as a primer pair (Pair primer).

1 is an electrophoretic photograph of a 500bp product amplified using primer pairs ITS4 and CO-1.

FIG. 2 shows the results of Southern hybridization with labeled plasmid pGM T-Easy Vector (Easy Vector) including the ITS I + II region cloned from Colletotrichum gloeosporioides. .

FIG. 3 is an electrophoretic photograph of products amplified from various genomic DNAs of Colletotrichum gloeosporioides.

Hereinafter, the present invention will be described in more detail.

[Production of Primer for Molecular Labeling Factor for Diagnosis of Anthrax]

In general, the amplification of a particular region of the fungal genome by PCR is very useful as a technique that provides much faster and more sensitive data than prior classification and identification of pathogens. In particular, the ITS and CO-1 regions of the fungus are highly conserved but highly conserved, and this region can be used to design primers specifically for detecting persimmon anthrax. In the last decade, a third classification method has been developed for basidiomycetes, particularly mushrooms and plant pathogens, based on physiological culture classification or molecular classification using molecular biological information. Among these various molecular biological attempts, phylogenetic classification based on ribosome RNA gene was considered as the most powerful classification method. The transcription units of the eukaryotic ribosomal RNA gene (ribosome DNA), including fungi, are 18S, 5.8S, and 28S rDNAs in turn separated by two internal transcribed spacers (ITS). Initially, comparisons of sequences against the 5.8S site were primarily used as a basis for classification, but because these sites are fairly short and highly conserved, with around 120 bases, nearly identical sequences among related species. ), There is a difficulty that cannot be used in the classification to distinguish between them. However, 18S and 28S rDNA have statistically reliable information and have been treated as a suitable means for discussing phylogeny because coexistence of high base, moderate and highly mutated parts. However, 18S and 28S are 1600bp and 3300-4800bp, respectively, and the determination of the entire nucleotide sequence takes a lot of time and effort, and partial sequencing is the base for comparison between genera having broader phylogenetic evolution. The lack of sequence pointed out the difficulty of using less information that could be used as a classification indicator.

By the way, ITS is about 500bp including 5.8S in the middle, which is easy to determine the base sequence, and it is suitable for taxonomic studies targeting a large number of species because it is less time-limited, and its molecular evolution rate is fast. Suitable for classifying species and genera that require diversity of sequences. In addition, since the 5.8S site is highly conserved, the ITS site can be compared with each other. With such abundant information and simplicity, it has been a useful tool for studying the relationship between species and genera within the same genus.

Therefore, the present invention amplifies the ITS I + 5.8S + ITS II region using ITS1 and ITS4 as a primer pair for PCR, wherein the amplified band is a pGM ™ -T easy vector. Cloning was performed, and the nucleotide sequence was decoded by an automatic sequencer. Then, the detected band was confirmed as Collettocomhum gloeosporioides by requesting the Gene Bank of the US to determine whether the genus Collettohumum, the cause of anthrax (Colletotrichum.spp). . Therefore, primers were prepared by primer 3 (Primer 3: Whitehead Institute / MIT Center for Genome Research, USA), a program for designing primers for PCR using the nucleotide sequences identified above. As a result, the molecular marker for diagnosing anthrax of the present invention uses ITS-4: tcctccgctt attgatatgc and CO-1: ataacccttt gtgaacatac as a pair of primers.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example: Preparation of Molecular Marker for Diagnosing Anthrax

After isolateing the colletolithum (Colletotrichum) causing anthrax on the persimmon tree, the strain was transferred to potato agar medium (PDA, Potato Dextrose Agar) and subcultured. To carry out the experiment, the strains incubated for 7 days in potato agar medium (PDA, Potato Dextrose Agar 20g / ℓ, sucrose 20g / ℓ, agar 17g / ℓ) were used for potato liquid medium (PDB, Potato Dextrose Broth 20g / ℓ, sucrose 20g / l) was inoculated using a cork-borer and incubated at 25 ° C for 15 days at 120 rpm.

In order to extract genomic DNA of Colletotrichum, Mycelia grown for 15 days in Potato Dextrose Broth (PDB) were used. After filtering the mycelia grown in potato liquid medium (PDB, Potato Dextrose Broth) with mineral wood cloth, the mycelium was lyophilized after washing 2-3 times with sterile water to remove the media components of the mycelia. Lyophilized hyphae was ground using liquid nitrogen and placed in a sterile 50ml tube, which was used while storing in a -70 ° C freezer. DNA extraction was performed by adding 10 ml (W / V) of lysis buffer (lysis buffer, 50 mM Tris-HCl, pH 8.0; 50 mM EDTA, pH 8.0; 3% Sodium Dodecyl Sulfate; 1% 2-mercaptoethanol) to 5 g of ground mycelia. After reacting in a constant temperature water bath for 1 hour, centrifugation at 3,000 rpm for 10 minutes, the supernatant was taken, and phenol / chloroform / isoamyl alcohol (25: 24: 1) and chloroform / isoamyl alcohol (Chloroform). / Isoamylalchol, 24: 1) was sequentially treated once, and then the supernatant was taken by centrifugation at 13,000 rpm for 10 minutes. The same amount of isoprophanol was added to the supernatant and stored in a -20 ° C freezer for at least 12 hours. The stored supernatant was centrifuged at 13,000 rpm for 10 minutes to precipitate DNA. Precipitated DNA was washed with 500 ml of 70% ethanol, washed, dried completely at room temperature, and added with TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH7.4) to which RNA digestase (RNase, 50 µl / ml) was added. After fully dissolved in 1 ml and stored for 1 hour at 37 ℃ (Fig. 1). The DNA was electrophoresed for 60 minutes using a 0.8% agarose gel at 100 V / cm 3 on a 0.5X buffer, and then the concentration was determined based on lambda DNA, which is already known on the UV box. Confirmed, the confirmed DNA was used by storing in the -20 ℃ freezer.

All fungi have ITS regions, which are quite conservative and highly mutated sites. In this study, we found that the use of these regions can design primers that specifically detect persimmon anthrax. A single band was obtained using primers of ITS5 and ITS4, which can partially amplify 18S and ITSI + 5.8S + ITSII.

So this band is called pGMCloning was performed using -T easy vector and sequencing was performed using an automatic sequencer. In order to know whether or not the sequence found in this experiment is choletotricom Gloeosporioides As a result of requesting to Gene Bank, it was identified as Colletotrichum gloeosporioides and Primer3, a program that designs primers for PCR from nucleotide sequence. Primer was prepared by the Whitehead Institute / MIT Center for Genome Research, USA.

Experimental Example 1: Identification of molecular markers using primers ITS-4 and CO-1

Pair primers were colletolicom gloeosporioides isolated from Gyeongju, Gimhae, Miryang, Changwon, and Changnyeong using ITS-4: tcctccgctt attgatatgc and CO-1: ataacccttt gtgaacatac. Not infected with, strained or semi-infected or infected persimmon leaves, and colletolecum hum gloeosporioides, or not infected with cholettotrichum gloeosporioides, Half-infectious or infectious persimmon, also, uninfected, semi-infected, or infected persimmon and pestalofthiopsis, lyzanto or solani, yuninia, phytophthora infestus, or fusidium PCR reactions were carried out on the Oxysis forum, as a result of the genus Mycosperella, Pestalofoptosis, Lychonia solani, Erwinia and Par. Saratov Torah inpe instance, or Fusarium oxy's forum was the fruit is amplified not happen uninfected Persimmon leaves, persimmon trees in the infected leaves and fruits showed that the band of about 500bp appears.

Experimental Example 2: Southern hybridization with plasmad pGM T-Easy vector containing ITS I + II region cloned from Colletotrichum gloeosporioides

PCR was performed using two primer pairs, CO-1 and ITS-4, which were loaded onto a 1% agarose gel. Soak the electrophoretic agarose gel in 0.25N HCl, shake it for 10 minutes, wash it with distilled water, store it again in dedensing buffer (0.5N NaOH, 1.5M NaCl) for 30 minutes, and then distilled water. Washed again with and stored in 0.4N NaOH solution until immediately before transfer. 0.4 N NaOH was transferred to a membrane for 16 hours using a transfer solution. Transfer membranes were washed using 6X SSC, and placed again between 3MM paper and dried at room temperature for 1 hour.

The ITS region of Colletotrichum gloeosporioides was cloned into pGEM ^(R) -T (Easy Vector, Promega, USA), and the pUC / Probes were prepared by PCR labeling using M13 forward primer (5'-CGCCAGGGTTTTCCCAGTCACGAC-3 ') and reverse primer (5'-TCACACAGGAAAAGCTA TGAC-3'). . PCR labeling reaction composition was 10 ng of plasmid DNA, M13 / pUC forward, reverse primer 0.5 μM, 10X buffer, 5 unit Taq polymerase, 0.1 mM dGTP, 100 μl of dCTP, dATP, 0.09mM dTTP, 1mM digoxigenin-dUTP (Boehringer Manhein, Germany) was used for 1 minute 30 seconds at 94 ° C, 1 minute 30 seconds at 50 ° C, and 2 minutes at 72 ° C. After repeating 25 times, the mixture was reacted at 72 ° C. for 4 minutes. After completion of the PCR reaction, 10 μl of the PCR product was taken and electrophoresed on a 1.5% agarose gel.

Transfer the transferred membrane to 60 ° C., add 20 ml of a standard hybridization solution (5X SSC, 0.02% SDS, 0.1% N-lauroylsarcosine, 1% blocking reagent) preheated to 60 ° C. After pre-hybridization, the existing buffer is removed, and 5 ml of a solution of 30 µl of a fully denatured probe and a standard hybridization solution is added again at 60 ° C. Hybridization was carried out for 12 hours.

2X SSC, 0.1% (w / v) SDS solution, 0.1X SSC, 0.1% (w / v) SDS solution to remove probes that do not adhere to the membrane after hybridization. The membranes were washed twice in 20 minutes each. The washed membrane was incubated for 30 minutes at room temperature in a solution diluted 1:10 of blocking stock solution with maleic acid buffer, and then again anti-dig-AP conjugate. The solution was incubated for 30 minutes at room temperature in a solution diluted 1: 5,000 with the buffer used in the whole procedure. Thereafter, the mixture was washed twice with a maleic acid buffer containing 0.3% (w / v) tween 20 for 15 minutes at room temperature to remove the non-stick conjugates. ) Was observed after staining with NBT / BCIP. As a result of using the ITS region of P-1 as a probe, Southern hybridization was performed using the ITS region of Co-1 as a probe. Riaoctonia , Fusarium , Pseudomonas , Ewinia, and diseased and healthy tissues were amplified by PCR and then transferred to a nylon membrane. Only the diseased tissue was able to detect a strong band (Fig. 2).

Experimental Example 3: Amplification of various amounts of Colletotrichum gloeosporioides genomic DNA

PCR was performed by diluting the concentration of anthrax pathogen to determine how sensitive the designed primer was. PCR reaction conditions were initially denatured at 95 ° C. for 5 minutes, followed by a final cycle of 35 cycles with 1 cycle at 95 ° C., 1 minute at 57 ° C., 1 minute at 72 ° C., and 10 at 72 ° C. After the reaction, the reaction was terminated. The amplified PCR product was subjected to electrophoresis for 2 hours on a 1% agarose gel using 0.5 × TBE (0.045M Tris-borate, 0.001M EDTA) buffer, followed by the appearance of DNA fragments developed in the UV box. Observed. As a result, a strong band was obtained at 10 ng and 100 pg, but a faint band was detected at 1 pg (FIG. 3). This shows that the minimum concentration of Colletotrichum can be detected up to 10 pg. Therefore, when using the prepared primer (primer) can detect up to a minimum concentration of 10pg, DNA extracted from the plant suspected of anthrax pathogens, and PCR using the PCR can determine the presence of anthrax pathogens in the persimmon tree Can be detected early, and the use of pesticides to control anthrax is expected to reduce costs.

As described above, the present invention relates to a molecular marker for diagnosing persimmon anthrax. Therefore, according to the present invention, since it is possible to detect anthrax pathogen infection up to a minimum concentration of 10 pg, it is not only effective to discriminate anthrax pathogen infection from persimmon trees, and as a result, for anthrax control Pesticides can be used to prevent overuse and reduce costs.

Claims (3)

DNA marker for diagnosing persimmon anthrax pathogens amplified using ITS-4 and CO-1 as a pair of primers. [Claim 2] The molecular marker for diagnosing persimmon anthrax in claim 1, wherein the ITS-4 has a nucleotide sequence of 1 in the sequence listing. [Claim 2] The molecular marker for diagnosing persimmon anthrax is characterized in that the base sequence is 2 in the following sequence list.