JP2923778B1 - Specific detection method of white root rot fungus - Google Patents

Abstract

Abstract: PROBLEM TO BE SOLVED: To provide a method for specific detection of fungus fungi. [Solution] White root rot fungus Rocerinia necatrix
(Rosellinia necatrix) a DNA containing a gene encoding a 5.8s ribosomal RNA, a DNA containing a gene encoding a 5.8s ribosomal RNA of a genus Rosellina other than white rot,
A DNA primer having a sequence specific to S. serrata, using the primers, 5.8s ribosomal RNA of S. serrata
A method for detecting a white root rot fungus, comprising amplifying a DNA containing a gene encoding the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to 5.8s of white root rot fungi
The present invention relates to a DNA containing a gene encoding ribosome RNA, a primer set for specifically and rapidly detecting and identifying the DNA, a method for detecting white rot, and a kit for detecting white rot.

[0002]

2. Description of the Related Art Roselinia necatrix
(Rosellinia necatrix) is a soil-borne fungus,
It mainly rots and kills the roots of trees, including fruit trees. White root rot fungi are widely distributed in temperate regions around the world, such as Europe, the Middle East, and the United States, and are one of the major factors inhibiting production of fruit trees such as pears, apples, grapes, and kiwifruits. White root rot fungi grow on plant residues in soil and infect roots, so it is extremely difficult to find out the site of infection and to take appropriate control.

[0003] In addition, white fungus fungi form a durable body called a pseudo-shell in the soil and can survive for several years. It is necessary to confirm that there is no fungal fungus. As a simple identification method for white root rot fungi, microscopy is performed by microscopic observation of hyphae and observing the pear-shaped swelling of the hyphal partition wall of the white root rot fungus.
[Journal of the Japan Society of Silk Science, 33: 161-166 (1964)], and a culture method using the property of causing the white root rot fungus to turn yellow when cultured in a medium containing aniline blue [Journal of the Japan Society of Silk Science, 47: 519-526]
(1978)].

[0004] However, in the case of the microscopic method, when the mycelium is young, no pear-shaped hypha is formed on the partition wall. In the case of the method, even in the filamentous fungi other than the fungus fungi other than the fungus fungi, there is a possibility that there is a bacterium that yellows the medium and there is a risk of causing erroneous detection. Difficult and difficult to obtain reliable results. Since there are few strains that have confirmed the spore morphology, which is the classification standard of white root rot fungi, up to now, regarding intraspecies differentiation, such as whether or not the one currently treated as white root rot is really the same species Has not been studied, and some of the isolates have very specific colony morphology.

[0005] Furthermore, among the genus and species of the same genus, there is a bacterium that hardly differs from the fungus of white root rot in the form of ascospores [Francis, S. et al.
M .: Sydowia 38: 75-86 (1985), Ezuka Akinori et al .: Tea Industry Research Report 40: 26-30 (1973)]. Therefore, it has been strongly desired to clarify the intraspecific differentiation of the fungus and to develop a means for specifically and quickly detecting and identifying the fungus.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to a DNA containing a gene encoding a 5.8s ribosomal RNA of white rot fungus,
It is an object of the present invention to provide a primer set for specifically and quickly detecting and identifying DNA, a method for detecting white rot, and a kit for detecting white rot.

[0007]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the fungus of white root rot fungi is
DNA containing the gene encoding the 5.8s ribosomal RNA was isolated, and a primer capable of rapidly and highly sensitively detecting white rot was successfully produced, thereby completing the present invention. That is, the present invention is a DNA comprising any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 4. Furthermore, the present invention relates to an oligonucleotide capable of hybridizing with at least a part of DNA having any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 4.

Further, the present invention provides a 5′-side primer capable of hybridizing with at least a part of any one of the nucleotide sequences represented by SEQ ID NOs: 1 to 4,
A primer set comprising a combination of a 3′-side primer capable of hybridizing with at least a part of any of the base sequences represented by Nos. 1 to 4. The 5′-side primer includes a primer represented by SEQ ID NO: 5, and the 3′-side primer includes a primer represented by SEQ ID NO: 6.

Further, the present invention provides a method for preparing 5.8 s ribosome RN of S. cerevisiae using any of the above primer sets.
It is a method for detecting white rot fungus, characterized by amplifying a gene containing DNA containing a gene encoding A. Furthermore, the present invention is a kit for detecting white rot fungus, which comprises any of the primer sets described above. Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The gene of the present invention is
DNA encoding 5.8s ribosomal RNA and its adjacent region (I
TS: Internal Transcribed Spacer)
It can be obtained as follows. 1. Isolation of 5.8S ribosomal RNA gene and its adjacent region (1) Preparation of genomic DNA Sources of genomic DNA include white root rot, Rosellinia arcuata, Rosellinia pepo, Roselinia Bunodes
llinia bunodes) and Rosellinia
quercina), Rosellinia aquil
a) Other fungi belonging to the genus Rosellina.

For example, the above strain is cultured in a liquid or solid medium such as a potato-dextrose medium, a bran medium and a malt medium at 25 ° C. for 4 to 14 days with shaking or standing. After the culture, the cells are collected by a filtration method, a centrifugation method, a scraping method, or the like. Next, genomic DNA can be extracted from the collected cells by a conventional method such as a method using a surfactant or a method of performing heat treatment.

(2) Amplification of 5.8s ribosomal RNA gene and its adjacent region by PCR In general, in the genome of fungi such as filamentous fungi including yeast of white root rot and fungi such as yeast, the region encoding the 5.8s ribosomal RNA and its adjacent region are , FIG. 1 is known. Oligonucleotide primers ITS1 (SEQ ID NO: 7) and ITS4 (SEQ ID NO: 8) from White et al. Capable of amplifying the fungal 5.8s ribosomal RNA gene and its flanking regions [White et al .: PCR Protocols, Academic Pres
s, San Diego, pp. 315 (1990)], and using the genomic DNA obtained in (1) above as a template, the polymerase chain reaction.
(Also referred to as PCR), the DNA of the present invention can be obtained.

(3) Determination of Nucleotide Sequence The obtained PCR fragment is subcloned into an appropriate vector such as pBlueScriptSK (+) (manufactured by STRATAGENE), pCR2.1 (manufactured by Invitrogen), and the nucleotide sequence is determined. . Or PCR
Perform direct sequencing using the fragments. The nucleotide sequence can be determined by the Maxam-Gilbert chemical modification method or M
It can be carried out by a known method such as the dideoxynucleotide chain termination method using 13 phages, but usually the sequence is determined using an automatic base sequencer (for example, 373A DNA sequencer manufactured by PERKIN-ELMER). Once the nucleotide sequence of the gene of the present invention is determined, it is then hybridized by chemical synthesis, by PCR using the cDNA or genomic DNA of the gene as a template, or by using a DNA fragment having the nucleotide sequence as a probe. As a result, the DNA of the present invention can be obtained.

(4) Synthesis of primers for detecting white rot fungi [SEQ ID NO: 1 and SEQ ID NO: 2]
SEQ ID NO: 3 and SEQ ID NO: 4 show the 5.8 s ribosomal RNA gene of the other genus Rosellina and the nucleotide sequence of the adjacent region thereof. These base sequences are compared to find a sequence characteristic of the fungus, and a synthetic oligonucleotide having the sequence can be used as a primer for detecting the fungus.

For example, the 5'-side primer and the 3'-side primer which can be used for the detection of fungal pathogens can be selected from any region of the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. . However, a region where a difference is observed between the strains belonging to the genus Rosellinia, a region having a high GC content, and a region where the primers do not hybridize is preferable.

For example, as the 5'-side primer, in SEQ ID NO: 1 or SEQ ID NO: 2, 12th to 25th, 65th to 126th, and
And a region consisting of at least 10 to 50 bases, more preferably 15 to 25 bases, such as the 150th to 174th bases. In addition, as the 3 ′ primer, 384th to 402th and 437 to 472th which show differences between species belonging to the genus Loceliner
The region consisting of at least 10 to 50 bases, more preferably 15 to 25 bases, such as the 1 st and 494 th to 510 th bases, is preferred. These primers can be used in any arbitrary combination.

The nucleotide sequences of the primers of the present invention are exemplified by SEQ ID NO: 5 and SEQ ID NO: 6, and hybridize with at least a part of a gene containing DNA consisting of any of the nucleotide sequences represented by SEQ ID NOS: 1 to 4. Oligonucleotides that can soy are also included in the primers of the present invention. The primer used in the present invention is not limited to these.

2. Detection of white root rot using the primer of the present invention (1) Preparation of test sample solution Preparation of a test sample solution that seems to contain white root rot, the root suspected to be infected with white root rot, DNA may be extracted from soil, cultured bacteria, mycelium trapped in plant branches and the like from the soil by a normal method such as a method using a surfactant or a method of performing heat treatment, which is then extracted with phenol, chloroform, ethanol, etc. And used as a sample solution for the specimen.

(2) Detection of white rot fungus by PCR Amplify the 5.8 s ribosomal RNA gene and its adjacent region using the S. blight fungus-specific primer described in, and fractionate the amplified fragment by agarose gel electrophoresis or polyacrylamide gel electrophoresis, and then run the gel. Stained with ethidium bromide, etc.
It can be performed by confirming the presence or absence of the amplified fragment under a UV lamp.

Amplification is performed using standard protocols [eg, Sambr
ook, J et al., Molecular Cloning, ColdSpring Harbor Labo
ratory Press (1989)], for example, the PCR reported by Saiki et al.
Method [Saiki, RK: Science, 239: 487-491 (1988),
61-274697], and its modifications and improvements are preferred. By these PCR methods and the like, a target DNA fragment can be specifically amplified from a small amount of a sample.

That is, the DNA sample is denatured to a single strand. Next, a 5 ′ primer complementary to the 5 ′ side of one strand of the target DNA sequence, a 3 ′ primer complementary to the 3 ′ side of the other strand, a DNA sequence used as a template, Using a reaction system containing nucleoside triphosphates (dATP, dCTP, dGTP, and dTTP or dUTP) and a DNA polymerase, the template DNA and the two primers are annealed. Next, a complementary strand is synthesized on each template from the DNA polymerase and the four deoxynucleoside triphosphates. Then, after the generated double strands are converted into single strands by thermal denaturation, the same annealing and complementary strand synthesis procedures as above are repeated using each strand as a template, so that only the portion between the primers is exponential. Can be increased.

Thus, the portion between the primers
By performing the above series of operations for N cycles (N = 20 to 40), a DNA fragment which is theoretically amplified by a factor of 2 ^N can be obtained. Since DNA polymerase can synthesize hundreds to thousands of bases per minute, one cycle can be performed in 5 to 7 minutes. Thus, for example, for 25 cycles of amplification,
It takes about 3 hours, in which case the efficiency of each cycle is 90
%, It can be amplified by (1 + 0.9) ^25, or about one million times.

As the DNA polymerase used in the PCR method or the like, it is preferable to use a polymerase having heat resistance at the DNA cleavage temperature, for example, Taq, Tth polymerase and the like. Therefore, it is almost unnecessary to replenish the enzyme each time heat denaturation is performed in each cycle, so that the enzyme can be easily and quickly performed by the PCR method or the like.

In the present invention, the DNA fragment to be amplified is
This is a DNA fragment containing the 5.8s ribosomal RNA gene and a part of the region adjacent to the 5.8s ribosomal RNA gene, which is possessed by the fungus fungi. In the PCR method and the like, the DNA sequence at the portion between the primers is amplified, and the size of the DNA fragment depends on the type of the primer used. In one example of the present invention, a 313 bp DNA fragment is amplified using the above primers. Next, the amplified DNA fragment is subjected to electrophoresis using agarose gel or acrylamide gel, and then stained using ethidium bromide or the like, and the presence or absence of the amplified fragment can be confirmed under a UV lamp.

In addition, by using the method of the present invention, it is difficult to discriminate by the microscopic method of observing the mycelium partition wall or the culture method using the aniline blue-containing medium, and the colony morphology is difficult to determine with the naked eye. It is also possible to accurately identify a white root rot fungus observed differently from a normal white root rot fungus. In the past, there was no difference in the form of ascospores, and it was considered to be the same species as the white root rot fungus, but it was classified as a different species in the taxonomy, belonging to the genus Rosellinia such as Rosellinia arcuata (Rosellinia arcuata) It was also shown that there was little evidence that filamentous fungi were considered to be different from the fungus fungi at the genetic level. Furthermore, the primer of the present invention can be used as a kit for detecting white rot fungus.

[0026]

The present invention will be described below in more detail with reference to Examples, but it should not be construed that the present invention is limited thereto. Example 1 Isolation of DNA Containing Gene Encoding 5.8s Ribosomal RNA of Rosellina sp. (1) Preparation of DNA from Rosellina sp. Rosellinia necatrix strains R-4- and R- twenty four-
The strains, Rosélinea pepo (R. pepo) CBS350.36, Rosélinea arcuata (R. arcuata) CBS347.29, and Rosélinea quercina (R. quercina) ATCC 36702, were placed at 25 ° C. in potato dextrose broth medium (Difco). , 4-
Cultured for 10 days. Next, the cells were collected by centrifugation at 4 ° C. and 12,000 × g for 3 minutes, and the cells were washed twice with sterile water.

The obtained cells with a wet weight of about 50 mg were added to 230 μl of a cell disruption buffer (50 mM Tris-HCl (pH 7.2), 50 mM EDTA,
1% SDS, 1% 2-mercaptoethanol), and the cell suspension was ground in a mortar using a pestle. Next, the microbial cell lysate was transferred to a microtube, and then incubated in a 65 ° C. water bath for 1 hour to obtain a microbial cell lysate.

[0028] TE-saturated phenol (1M
230 μl of an equal mixture of 0.1% 8-hydroxyquinoline (saturated with Tris-HCl (pH 8.0) saturated with 0.1% 8-hydroxyquinoline) and chloroform / isoamyl alcohol (24: 1) is added, mixed vigorously, and then mixed at 12,000 × g for 10 minutes. Centrifuge. After centrifugation,
Transfer the upper layer to a new microtube, add 1/10 volume of 3M sodium acetate and 54/100 volume of isopropanol,
It was left standing for about 30 minutes.

Next, DNA was pelleted by centrifugation at 15,000 xg for 10 minutes at 4 ° C. 70% of the resulting DNA precipitate
After rinsing with cold ethanol, the mixture was centrifuged again at 4 ° C. and 15,000 × g for 10 minutes. After drying the DNA precipitate, 300 μl
It was dissolved in a TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), heat-denatured at 95 ° C. for 2 minutes, and ice-cooled and stored frozen as a DNA sample solution.

(2) Preparation of primers Primers were prepared according to the method of White et al. [White et al .: PCR P
rotocols, Academic Press, San Diego, pp. 315 (199
0)], the ITS containing the fungal 5.8S ribosomal RNA gene
Primers for amplifying the region [White et al .: PCR P
rotocols, Academic Press, San Diego, pp. 315 (199
0)]. That is, the 5 'sense primer sequence
5'-TCCGTAGGTGAACCTG (also called primer ITS1)
CGG-3 '(SEQ ID NO: 7) was designated as 5'-TCCTCCGCTTATTG as a 3' antisense primer sequence (also referred to as primer ITS4).
ATATGC-3 '(SEQ ID NO: 8) was synthesized. The synthetic oligonucleotide was chemically synthesized using a fully automatic DNA synthesizer.

(3) Amplification of DNA containing gene encoding 5.8 s ribosomal RNA of Locerenia sp. By PCR The DNA prepared in (1) above was used as a template, and the primer prepared in (2) above was used as a primer. PCR was performed using ITS1 and primer ITS4. The composition of the PCR reaction solution is as follows.

DNA solution 1.0 μl 10 × PCR buffer (manufactured by TOYOBO) 2.5 μl 2 mM dNTPs 2.5 μl 25 mM MgCl ₂ 1.5 μl 2 μM sense primer (ITS1) 5.0 μl 2 μM antisense primer (ITS4) 5.0 μl 5 U / μl Taq polymerase 0.15μl distilled water 7.35μl total volume 25.0μl

The PCR was performed using a thermal cycler (manufactured by ASTEC,
Heat denaturation at 94 ° C for 1 minute and 55 ° C for 2 minutes.
The conditions of annealing for one minute and extension reaction at 72 ° C. for one minute were defined as one cycle, and 30 cycles were performed. The obtained PCR product was subjected to a fluorescence automatic D using a dye terminator cycle sequencing FS Ready Reaction Kit (ABI).
Analysis was performed using an NA sequencer (ABI, model 377).

As a result, as shown in FIG. 2 and FIG. 3, the sequence derived from the Roselinia arcauce CBS347.29 and the sequence derived from the Roselinia necatrix R-24-strain had a total nucleotide sequence of 549 bp determined. Matched (SEQ ID NO: 2). Sequence from Roseliner Necatrix R-4-strain
(SEQ ID NO: 1) is for Roseliner Necatrix R-24-
It was different from the strain-derived sequence (SEQ ID NO: 2) by only one base at the 52nd base. On the other hand, the sequences derived from Rosellinia pepo CBS350.36 (SEQ ID NO: 3) and Rosenella quercina ATCC36702
(SEQ ID NO: 4) is Roserine Necrotrix R-4-
The sequence was slightly different from the sequences from the strain, the Roselinia Necatrix R-24-strain, and the Roselinia Arcuata CBS347.29.

[Example 2] 5.8s ribosome of white root rot fungus
Amplification of DNA containing RNA-encoding DNA by PCR and detection of DNA fragments. (1) Preparation of DNA of white root rot fungus containing ITS region containing 5.8s ribosomal RNA gene. 49 strains of white root rot (Rosellinia necatrix). DNA of (Table 1)
Was prepared in the same procedure as in Example 1.

[0036]

[Table 1]

(2) Selection and Synthesis of Oligonucleotide Primers The white rot fungus (Rosellinia necatrix) decoded in Example 1
R-4-share, R-24-share), Roselinia Pepo CBS350.36,
Roseliner Arcuate CBS347.29, Roseliner
Compare and examine the nucleotide sequence of the 5.8s ribosomal RNA gene of Quercina ATCC36702 and its adjacent regions, and select a nucleotide sequence region that does not differ between species, does not contain a region with high GC content, and does not hybridize with primers. (FIG. 2 and FIG. 3, shaded area). That is, as a sense primer, 5′-GCATTGAATTCTGAACACA-3 ′ (SEQ ID NO: 5)
5'-CCATAGGCGAGATGAGA as antisense primer
AATC-3 '(SEQ ID NO: 6) was selected. The above primers were chemically synthesized according to a conventional method.

(3) Amplification of DNA fragment containing 5.8s ribosomal RNA gene of white rot fungus and a part of the adjacent region thereof The DNA prepared in the above (1) was amplified by PCR in the same manner as in Example 1. And amplified. However, the primer synthesized in the above (2) was used. (4) Detection of Amplified DNA Fragment 3 μl of the reaction solution containing the amplified DNA fragment obtained by PCR in the above step (3) was subjected to electrophoresis on a 2% agarose gel together with a DNA size marker, followed by ethidium bromide staining. FIG. 4 shows a part of the result. As shown in FIG. 4, a specific DNA fragment of 313 base pairs was observed in all of the 49 strains tested regardless of the host, the isolation site, and the colony morphology.

Example 3 Assay for Crossing Ability (1) Assay for Crossing Ability Using Primers of the Present Invention Example 1 DNA prepared in Step A and Rosellinia bunodes CBS 347.36 and Rosellinia Aquila MAFF4200
Using DNA prepared in the same manner as in Example 1 from 64,
According to the same procedure as in Example 2, of the filamentous fungi belonging to the genus Locerenia, five species having root pathogenicity were tested for crossability. The result is shown in FIG.

As shown in FIG. 5, when the PCR was carried out using the specific primers, it was confirmed that the specific primers were the same species as R. necatrix and Rosa nectrix. , SMS
ydowia 38: 75-86 (1985), Akinori Ezuka et al .: Tea Industry Report 4
0: 26-30 (1973)] alone, and no amplified fragment was detected from the other four species. From this, it was confirmed that this primer did not react even between closely related species, and was a primer that specifically detected white rot fungus.

(2) Detection of white root rot bacteria using the primers of the present invention According to the same procedure as in Example 2, 29 strains were randomly selected from strains isolated from pear roots highly sensitive to white root rot. , Crossover test was performed. FIG. 6 shows a part of the result. In the PCR using the primers, a specific DNA fragment of 313 base pairs was amplified only from the fungus fungus, but not from other strains.

[0042]

Industrial Applicability According to the present invention, a gene, a primer, and a gene for specific and rapid detection and identification of a fungus of white root rot are provided.
A detection method is provided.

[0043]

[Sequence list] SEQ ID NO: 1 Sequence length: 549 Sequence type: Number of nucleic acid chains: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Rosellinia necatrix Strain name: R-4- SEQ AGGGATCATT AAAGAGTTCT ATAACTCCCA AAACCCATGT GAACATACCA CACGTTGCCT 60 CGGCAGGTCG CGTCCTACCC CGAAGTGCCC TACCCTGTTA GGGCCTACCC GGTGGGCGCG 120 GGCCAACCTG CCGGCGGCCC ACGAAACTCT GTTTAGCATT GAATTCTGAA CACATAACTA 180 AATAAGTTAA AACTTTCAAC AACGGATCTC TTGGTTCTGG CATCGATGAA GAACGCAGCG 240 AAATGCGATA AGTAATGTGA ATTGCAGAAT TCAGTGAATC ATCGAATCTT TGAACGCACA 300 TTGCGCCCAT TAGTATTCTA GTGGGCATGC CTGTTCGAGC GTCATTTCAA CCCTTAAGCC 360 CCTGTTGCTT AGTGTTGGGG GCCTGCAGCG CCTGCTGCAG CCCCTCGAAG TCAGTGGCGG 420 AGTCGGTCAC ACACTCTAGA CGTAGTAGAT TTCTCATCTC GCCTATGGTT GTGCCGGTCC 480 CCTGCCGTAA AACACCCCCC TATACCAAAG GTTGACCTCG GATCAGGTAG GAATACCCGC 540 TGAACTTAA 549

SEQ ID NO: 2 Sequence length: 549 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Rosellinia necatrix Strain name: R-24- Origin organism: Rosellinia arcuata strain name: CBS347.29 sequence AGGGATCATT AAAGAGTTCT ATAACTCCCA AAACCCATGT GAACATACCA CGCGTTGCCT 60 CGGCAGGTCG CGTCCTACCC CGAAGTGCCC TACCCTGTTA GGGCCTACCC GGTGGGCGCG 120 GGCCAACCTG CCGGCGGCCC ACGAAACTCT GTTTAGCATT GAATTCTGAA CACATAACTA 180 AATAAGTTAA AACTTTCAAC AACGGATCTC TTGGTTCTGG CATCGATGAA GAACGCAGCG 240 AAATGCGATA AGTAATGTGA ATTGCAGAAT TCAGTGAATC ATCGAATCTT TGAACGCACA 300 TTGCGCCCAT TAGTATTCTA GTGGGCATGC CTGTTCGAGC GTCATTTCAA CCCTTAAGCC 360 CCTGTTGCTT AGTGTTGGGG GCCTGCAGCG CCTGCTGCAG CCCCTCGAAG TCAGTGGCGG 420 AGTCGGTCAC ACACTCTAGA CGTAGTAGAT TTCTCATCTC GCCTATGGTT GTGCCGGTCC 480 CCTGCCGTAA AACACCCCCC TATACCAGATCAG TAGGATCAGGATCACC TAGGATCGATCACC

SEQ ID NO: 3 Sequence length: 537 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Rosellinia pepo Strain name: CBS350.36 Sequence AGGGATCATT ACAGAGCGCA CAAGCTCCCA AACCCACTGT GAACATACCA CACGTTGCCT 60 CGGCGGGGGG CTTTCAGGGC CCGAGAAGGG CCCCGAAGCG AGCCCGCCGG CGGCCCACGA 120 AACTCCGTCT AGCCATTGAT ATCTCTGAAC TTGATAACTG AATCAGTTAA AACTTTCAAC 180 AACGGATCTC TTGGTTCTGG CATCGATGAA GAACGCAGCG AAATGCGATA AGTAATGTGA 240 ATTGCAGAAT TCAGTGAATC ATCGAATCTT TGAACGCACA TTGCGCCCGC TAGTATTCTA 300 GCGGGCATGC CTGTTCGAGC GTCATTTCAA CCCTTAAGCC CCAGCTGCTT AGTGTTGGGG 360 GCCCGCGGCG CGCCACACTG CCCGCGGCCC CTCGAAGTTA GTGGCGGAGT CGGTTGCCCA 420 CCCCAGGCGT AGTAGATCTC TCGTCTCGCC TGCGGCGGCG CCGGTCCCCT GCCGTAAAAC 480 CCCCCATCTA TCCAAAAGGT TGACCTCGGA TCAGGTAGGA ATACCCGCTG AACTTAA 537

SEQ ID NO: 4 Sequence length: 553 Sequence type: Number of nucleic acid strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Rosellinia quercina Strain name: ATCC36702 Sequence AGGGATCATT ACAGAGTTTA TCTTAACTCC CAAACCCCAT GTGAACATAC CTTACGTTGC 60 CTCGGCAGGT CGCGTCGTAC CCGGGAGGGC CCTACCCTGT AGGGCATGAC CTGGTGCGCG 120 CGGGTAACCC TGCCGGCGGC CCCTGAAACT CTGTTTTTTA CATTAGAATT CTGAATCTAT 180 AACTAAATAA GTTAAAACTT TCAACAACGG ATCTCTTGGT TCTGGCATCG ATGAAGAACG 240 CAGCGAAATG CGATAAGTAA TGTGAATTGC AGAATTCAGT GAATCATCGA ATCTTTGAAC 300 GCACATTGCG CCCATTAGTA TTCTATTGGG CATGCCTGTT CGAGCGTCAT TTCAACCCTC 360 AAGCCCCCGT TGCTTGGTGT TGGGAGCCTA CGGCGACGTA GCTCCTCAAA GTTAGTGGCG 420 GAGTCGGCTC GCACTCTAGA CGTAGTAGAA TCTTTTTATC TCGCCTGTAG TGTGTGCCGG 480 TCCCCTGCCG TAAAACCCCC CCAATTATCA AAAGGTTGAC CTCGGATCAG GTAGGAATAC 540 CCGCTGAACT TAA 553

SEQ ID NO: 5 Sequence length: 19 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence GCATTGAATT CTGAACACA 19

SEQ ID NO: 6 Sequence length: 21 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence CCATAGGCGA GATGAGAAAT C 21

SEQ ID NO: 7 Sequence length: 19 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCCGTAGGTG AACCTGCGG 19

SEQ ID NO: 8 Sequence length: 20 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence TCCTCCGCTT ATTGATATGC 20

[Brief description of the drawings]

FIG. 1 is a diagram showing a region encoding a 5.8s ribosomal RNA on a fungal genomic DNA and a region adjacent thereto.

[Fig. 2] Roseliner pepo (R. pepo), Rosenera necatrix (R. necatrix) R-4-strain, Roseriner-necatrix R-24-strain, Roseneria arcuata
(R. arcuata) and R. querci (R. querci)
FIG. 7B is a diagram showing the nucleotide sequence of the ITS region containing the 5.8s ribosomal RNA gene of na) (excluding the primer sites of ITS1 and ITS4) and their alignment. The shaded area is the site used as a primer for specific detection this time. Each sequence further continues to the corresponding sequence in FIG.

[Figure 3] Roseliner pepo (R. pepo), Roseneria necatrix (R. necatrix) R-4-strain, Roseriner-necatrix R-24-strain, Roseneria arcuata
(R. arcuata) and R. querci (R. querci)
FIG. 7B is a diagram showing the nucleotide sequence of the ITS region containing the 5.8s ribosomal RNA gene of na) (excluding the primer sites of ITS1 and ITS4) and their alignment. The shaded area is the site used as a primer for specific detection this time. Each array in the figure is a continuation of the corresponding array in FIG.

FIG. 4 is an electrophoretic photograph showing the result of amplifying a specific DNA fragment from white rot fungus with a primer developed according to the present invention.

FIG. 5 shows the results of using the primers of the present invention that are specific to white root rot fungi.
4 is an electrophoretic photograph showing the results of amplification by PCR.

FIG. 6 is an electrophoretic photograph showing the results of amplification using a primer derived from filamentous fungi of the present invention using a DNA derived from a filamentous fungus isolated from a pear root as a template.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int. Cl. ⁶ Identification code FI C12R 1: 645) (56) References Mylogia, Vol. 87 (1995) p. 72-83 Mol. Phylogenet. Evol. , Vol. 7 (1997) p. 103-116 Mol. Plant Microbe Interact. , Vol. 8 (1995) p. 709-716 (58) Fields investigated (Int. Cl. ⁶ , DB name) C12N 15/09 ZNA C12Q 1/68 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (3)

(57) [Claims] (1) a 5′-side primer represented by SEQ ID NO: 5;
Including the 3′-side primer represented by SEQ ID NO: 6 and SEQ ID NO: 6
Marset. 2. The 5′-side primer represented by SEQ ID NO: 5
Including the 3′-side primer represented by SEQ ID NO: 6 and SEQ ID NO: 6
5.8s ribosomal RNA of white rot fungus using merset
Amplifying DNA containing a gene encoding
A method for detecting white root rot fungus. 3. The 5′-side primer represented by SEQ ID NO: 5
Including the 3′-side primer represented by SEQ ID NO: 6 and SEQ ID NO: 6
A kit for detecting white rot fungus, including merset.