JPH10234399A - Sequence of nucleic acid for detection of fungus belonging to genus pythium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new sequence of nucleic acid comprising an oligonucleotide for detecting and identifying a fungus belonging to the genus Pythium, containing a specific base sequence or a base sequence which is complementary to the sequence and capable of simply, rapidly and accurately detecting and identifying a soil-born fungus. SOLUTION: This new oligonucleotide comprises either one base sequence of formula I to formula VIII or a base sequence which is complementary to the base sequence and is used for detecting and identifying fungus belonging to the genus Pythium and is useful as a probe, etc., for rapidly, simply and accurately detecting and identifying a fungus belonging to the genus Pythium and causing soilborne disease to crops in good sensitivity. The oligonucleotide is obtained by extracting DNA according to the conventional method from a fungus belonging to the genus Pythium, e.g. Pythium myriotylum KPMS, carrying out PCR by a primer prepared by using 18 SrRNA gene sequence of other fungus resistered in gene sequence data bank as a reference and selecting a base sequence part which is specific to the fungus of the genus Pythim from the resultant 18SrRNA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to oligonucleotides which are effective for detecting and identifying soil-borne pathogenic fungi.
More specifically, a fungus belonging to the genus Picium, which is a pathogenic fungus that causes soil disease in crops, can be rapidly, simply, and accurately detected using a sequence of 18S ribosomal RNA (hereinafter referred to as "18S rRNA") unique to the genus. The present invention relates to an oligonucleotide for well detecting and identifying, and a method for detecting and identifying a fungus belonging to the genus Picium using the oligonucleotide.

[0002]

BACKGROUND OF THE INVENTION Control of crop soil pathogens in the agricultural field is an important issue. In particular, soil-borne fungi (molds) are the biggest cause of continuous cropping failure in upland farming. Common methods for detecting and identifying pathogens of crop soil include direct microscopic observation of the diseased part, and separation and culture of the pathogenic bacteria from the diseased part (Takiro Ichitani: Antibacterial and antifungal, 20, 107 -116 (1992)), it is said that these methods require a long time to determine, low detection sensitivity, and at least require phytopathological expertise and techniques. There are drawbacks. On the other hand, using an antibody against soil pathogens, an immunological method (Schots et al., Modern Assays for Plant Pathogen
ic Fungi: Identification, Detection and Quantifica
tion: CAB INERNATIONAL (1994)) is simple and can shorten the determination time, but has the disadvantage that the specificity of the reaction itself is limited. Therefore, in order to carry out appropriate disease control measures at an early stage, the development of a diagnostic / identification kit that can easily and quickly identify the soil pathogenic bacteria causing the outbreak is desired in agricultural fields.

By the way, among the pathogens of microorganisms in crop soil, the genus Pycium invades seedlings having a weak defense mechanism of a wide variety of crops, young roots at the root tips or roots in senescence, and causes seedling wilt and root rot. It is a soil pathogen with a broad host range and distribution range. It is known that the bacterium of the genus Pythium has a property of causing a crop disease explosively in a soil with a high water content because of its propagating property by water. In addition, the genus Pycium survives in the soil as a durable body called oospore, which is resistant to heat, dryness, chemical substances, or microorganisms, and germinates and necrotices roots that do not grow. It is known to have the property of causing mainly. Furthermore, it is also known that even when there is no cultivated crop, it has a property that it grows explosively immediately after application of fresh organic matter such as straw or green manure containing a large amount of water-soluble sugar.

[0004] Therefore, it has been desired to develop a simple, rapid, accurate and highly sensitive detection or identification method for the bacterium of the genus Pycium, especially among the pathogenic microorganisms of crop soil microorganisms.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel oligonucleotide which can be used for simply, quickly and accurately detecting or identifying a bacterium belonging to the genus Picium, which is a soil-borne pathogenic fungus. And

[0006]

Means for Solving the Problems The present inventors utilize the affinity between complementary nucleotide sequences to achieve a quicker method than conventional methods of microscopic observation of diseased parts and separation and culture of pathogenic bacteria from diseased parts. It was considered that the detection or identification of a genus Picium can be performed with high sensitivity and with higher specificity than a conventional method using an antigen-antibody reaction. In particular, we have:
18S has the characteristic that there is a region where the characteristic nucleotide sequence is highly conserved among genera due to slow evolution
Focusing on the rRNA gene, select a base sequence specific to the genus Picium out of the base sequences contained in the gene, and it may be possible to efficiently detect or identify the genus Picium by using the sequence. Thought.

The present inventors have conducted intensive studies based on this idea, and as a result, isolated the 18S rRNA gene from the genus Picium, determined the entire nucleotide sequence thereof, and determined the 18S rRNA gene of a bacterium belonging to another genus.
By comparing with the base sequence of rRNA, we succeeded in selecting and specifying a base sequence specific to the genus Picium. Furthermore, the present inventors actually prepared probes and primers based on the base sequence specific to this genus Picium,
PCR that allows simple and rapid detection using these nucleic acid molecules
Of the genus Pycium was detected by the method of hybridization and the hybridization method. As a result, the present inventors have found that the genus Picium can be detected specifically and with high sensitivity by using these nucleic acid molecules, and have completed the present invention.

As for fungi such as Candida and Cryptococcus, 18
An oligonucleotide for detection that hybridizes with the base sequence of the S rRNA gene has been disclosed (JP-A-8-8925).
4), No application to Picium spp. Is known.

That is, the present invention relates to a nucleic acid molecule capable of detecting and identifying a genus Picium simply, rapidly, specifically, and with high sensitivity, and a method for detecting a genus Picium using the nucleic acid molecule. More specifically, the present invention relates to (1) detecting or identifying a fungus belonging to the genus Picium, comprising a nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8, or a nucleotide sequence complementary thereto. For example, (2) a probe comprising the oligonucleotide described in (1), and (3) a primer comprising the oligonucleotide described in (1).

Further, the present invention provides a method for detecting or identifying a genus Picium, which comprises using the probe according to (4) or (2) or the nucleic acid primer according to (3).
About.

In the present invention, the genus Picium is defined as
Fungi belonging to the genus Picium, for example, Pythium acanthophoron (Pythium acanthophoron), Pythium afertile (Pythium afertile), Pythium aphanidermatium (Pythiumaphnidermatum), Pythium aristosporum (Pythium aristosporum), Pythium carolinianum,
Pythium conidiophoru
m), Pythium debaryanu
m), Pythium deliense, Pythium dissimile, Pythium dissotocum, Pythium echinulatum, Pythium echinulatum, Pythium echinulatum
Elongatium (Pythium elongatum)
Graminicola (Pythium graminicola), Pythium hydnosporum (Pythium hydnosporum),
Pythium inflatum, Pythium intermedium, Pythium inflatum
Pygium irregulare, Pythium iwayamai, Pythium myriotylum, Pythium rostratum, Pythium spinostam
um spinosum), Pythium s.
plendens), Pythium paddicu
m), Pythium sylvaticu
m), Pythium torulosum, Pythium ultimum, Pythium vanterpooli, and Pythium vexans.

The oligonucleotide of the present invention has a DN
Any of A or RNA, in the case of RNA,
In the description of the sequence in this specification, a thymidine residue (T) is to be read as a uridine residue (U).

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The oligonucleotide of the present invention comprises
In order to enable specific and highly sensitive detection, it is preferable that the nucleotide sequence be as specific as possible to the genus Picium as high as possible. Such sequences include, for example, SEQ ID NO:
Oligonucleotides containing any one of base sequences 1 to 8 or a base sequence complementary thereto are included.

The oligonucleotide of the present invention may have one or more bases substituted for 18S rRNA naturally possessed by the genus Picium, as long as it has a specific affinity for the 18S rRNA gene of the genus Picium. It may contain a deleted or added base sequence.

The origin of the oligonucleotide of the present invention is as follows:
There is no particular limitation. The oligonucleotide of the present invention can be prepared mainly by a chemical synthesis method.

The oligonucleotide of the present invention is used as a hybridization probe for detecting or identifying the 18S rRNA gene of the genus Picium. When the oligonucleotide of the present invention is used as a probe, the oligonucleotide is usually labeled. As the label of the oligonucleotide, for example, non-radioactive substances such as biotin, digoxigenin, enzymes, fluorescent substances, luminescent substances, or radioisotopes are used, but are not limited thereto. Labeling methods include 3′-tailing, 3 ′ end labeling, and 5 ′ end labeling. When the present probe is used to detect a sequence amplified by PCR or the like, a probe within the range of the amplified sequence is selected.

The detection or identification of a bacterium belonging to the genus Picium using the probe of the present invention is performed by a known method, for example, Southern hybridization (Southern, EM: J. Mol. Biol.,
98,503-517 (1975)), Northern hybridization (Thomas, PS: Proc. Natl. Acad. Sci. USA, 77,
5201 (1980)), dot hybridization (Vrie
s, MV et al: Gene, 50, 313-320 (1986)).

The oligonucleotide of the present invention is also useful as a primer for nucleic acid amplification. The primer of the present invention can be prepared by a chemical synthesis method such as the phosphoramidite method or the like, which is automatically synthesized by an automatic DNA synthesizer.

The detection or identification of a bacterium belonging to the genus Picium using the primers of the present invention is performed by amplifying a nucleic acid by a known method, for example, by amplifying DNA in a sample by performing PCR using the primers of the present invention. The method can be performed by detecting the amplified DNA. When the sample is RNA, the RNA is amplified as cDNA by using reverse transcriptase in the first reaction (RT-PCR). The amplified DNA can be easily detected by staining with ethidium bromide after agarose gel electrophoresis. Alternatively, it may be detected by a hybridization method using the probe of the present invention.

The test sample derived from the genus Picium used in the above-mentioned detection or identification method includes diseased tissue of a crop, planted soil, a parasitic animal, a culture, or DNA, RNA, Includes amplified DNA and the like. DNA and RNA were prepared by cetyltrimethylammonium bromide (CATB) and guanidine thiocyanate treatment, followed by phenol extraction, chloroform extraction, and phenol extraction.
It can be performed by a method such as chloroform extraction and ultracentrifugation.

The detection or identification method of the present invention can be used, for example, to avoid disease damage such as continuous cropping failure, to take early control measures, and to select optimal pesticides to be used.

[0022]

EXAMPLES The present invention will be specifically described below based on examples, but the examples do not limit the present invention.

[Example 1] 18S rR specific to the genus Picium
Selection and identification of nucleotide sequence in NA gene Pysium myriotilum K
PMS (Pythium myriotylum KPMS), Pythium spinosum OPA-1 and Fusarium oxysporum F. sp. Fragariae (Fusarium oxysporum f.sp.fragaria)
e) and Fusarium solani f. sp. phaseoli, Rhizoctonia solani AG-1 Cs-Gi (R
hizoctonia solani AG-1 Cs-Gi) and Rhizoctonia solani
Solanie AG-2-2 OPR-4 (Rhizoctonia solani AG-2-2 O
PR-4) was selected. Murry from these strains
(Nucleic Acids Res., 8: 4321-4325 (1980))
DNA was extracted according to the CTAB (Cetyltrimethylammonium bromide) method described in
enBank) registered with Candida albicans (Can
dida albicans) primers 5′-CTGGTTGATYCTGCCAGT-3 ′ (SEQ ID NO: 14) and 5′-CYGCAGGTTCACCTACRG-3 ′ (SEQ ID NO: 2)
By performing PCR using 9), the 18S
A ribosomal DNA (hereinafter referred to as 18S rDNA) sequence region was amplified. After cloning this into E. coli and isolating it as a recombinant plasmid, the Candida albicans 18S
16 types of primers 5'-CTGGTTGATYCTGCCAGT-3 '(SEQ ID NO: 14) prepared with reference to the rRNA gene sequence, 5'-GAA
ACTGCGAATGGCTCATT-3 '(SEQ ID NO: 15), 5'-AGGGYTCGA
YYCCGGAGA-3 '(SEQ ID NO: 16), 5'-CGCGGTAATTCCAGCTC
CA-3 '(SEQ ID NO: 17), 5'-TTRATCAAGAACGAAAGT-3'
(SEQ ID NO: 18), 5'-AATTTGACTCAACACGGG-3 '(SEQ ID NO: 19), 5'-ATAACAGGTCTGTGATGCCC-3' (SEQ ID NO: 2)
0), 5'-TTTGYACACACCGCCCGTCG-3 '(SEQ ID NO: 21)
(The above eight types are sense primers: R is A or G, Y is C or
T) and 5'-AATGAGCCATTCGCAGTTTC-3 '(SEQ ID NO: 22),
5'-TCTCCGGRRTCGARCCT-3 '(SEQ ID NO: 23), 5'-ATTACC
GCGGCTGCTGGC-3 '(SEQ ID NO: 24), 5'-TTGGYRAATGCTTT
CGC-3 '(SEQ ID NO: 25), 5'-CCGTCAATTYYTTTRAGTTT-3'
(SEQ ID NO: 26), 5'-GGGCATCACAGACCTGTTAT-3 '(SEQ ID NO: 27), 5'-GACGGGCGGTGTGTRC-3' (SEQ ID NO: 2)
8), using 5′-CYGCAGGTTCACCTACRG-3 ′ (SEQ ID NO: 29) (the above eight kinds are antisense primers: R represents A or G, Y represents C or T), and all bases of 18S rDNA of the above strain The sequence was determined (FIGS. 1-9). In particular, the entire gene sequence of Pythium myriotylum KPMS (Pythium myriotylum KPMS) is shown in SEQ ID NO: 12, and Pythium spinosam OPA-1 (Pythiu
SEQ ID NO: 13 shows the entire gene sequence of m spinosum OPA-1).

By comparing the total nucleotide sequences of the 18S rDNA of the 6 strains of the above 3 genera and juxtaposing them (FIGS. 1 to 9), the sequences of the two species belonging to the genus Pysium are common to the two strains of Picium and greatly differ from those of the other two species. A unique sequence region was found. In FIGS. 1 to 9, the portion surrounded by a square is a sequence homologous among the six bacteria.
These characteristic sequences were compared to the 18S r registered in the fungal gene data bank (GenBank) determined to date.
Compared to the RNA gene, it was found that the nucleotide sequences described in SEQ ID NOs: 1 to 8 were nucleotide sequences specific to the genus Picium. The strain names of the GenBank sequences used for the search are listed below. The information in parentheses indicates information such as the GenBank registration number, the deposit number, and the gene sequence region. Leptosphaeria bicolor A (LBU04202 Leptosphaeria bicolor A
TCC 4265218S ribosomal RNA gene), Leptosphaeria doliolum (LDU04205 Leptosphaeria doliolum)
IMI 199775 18S ribosomal RNA gene, LMU04233 Leptosphaeria maculan
s inernal transcribed spacer 1 and 18S ribosomal R
NA gene), Leptosphaeria microscopica (LMU)
04235 Leptosphaeria microscopica ATCC 38151 18S ri
bosomal RNA gene), Leptosphaeria maculans (LMU04238 Leptosphaeria maculans 18S ribosomal RN)
A gene), Pleospora harvalam (PHU05201 Pleos)
pora herbarum DAOM 150679 nuclear 18S ribosomal RNA
gene), Saccharomyces cerevisiae (CU02969 Sac)
charomyces cerevisiae nucleocytoplasmic 18S riboso
mal RNAgene), Septoria Nodrum (SNU04236 Sept)
oria nodorum DAOM 215173 18Sribosomal RNA gene),
Dipodascopsis Uninucleata (U00969 Dipodas)
copsis uninucleata 18S ribosomal RNA gene), U00970 Eurotium rubrum 18S rib
osomal RNA gene), Tafrina deformans (U0097)
1 Taphrina deformans 18S ribosomal RNA gene, U00974 Endomyces ge
otrichum 18S ribosomal RNA gene)
Rudis (U00975 Pleospora rudis 18S ribosomal RNA
gene), Tilletia caries (U00972 Tilletia c
aries 18S ribosomal RNA gene), Ustillago hordei (U00973 Ustilago hordei 18S ribosomal RNA gene)
e), Tremella globospora (U00976 Tremella glo
bospora 18S ribosomal RNA gene), U00977 Tremella moriformis 18S ribosom
al RNA gene), Alternaria alternanate (AAU05194)
Alternaria alternata AA6 nuclear 18S ribosomal RNA
gene), Alternaria brassicae (ABU05196 Altern)
aria brassicae AB11 nuclear 18S ribosomal RNA gen
e), Alternaria Brush Sicola (ABU05197 Alternar)
ia brassicicola ABc2 nuclear 18Sribosomal RNA gen
e) Alternaria r (ARU05199 Alternaria r)
aphani AR6 nuclear 18S ribosomal RNA gene), AUXRGEA Auxarthron zuf
fianum 18S ribosomal RNA (18S rRNA) gene), BMRRGE Blumeria graminis 18S rib
osomal RNA gene), Condor Marvinella (KONRDNA2)
Kondoa malvinella small subunit ribosomal DNA),
MBCRGEA Malbranche
a albolutea 18Sribosomal RNA (18S rRNA) gene), MBRCGEB Malbranche
a dendritica 18S ribosomal RNA (18S rRNA) gene),
MBCRGEC Malbranc
hea filamentosa 18S ribosomalRNA (18S rRNA) gen
e), MBCRGED Malbranch
ea gypsea 18S ribosomal RNA (18S rRNA) gene), Spolidioboras johnsonii (SOIRG18SA Sporidio)
bolus johnsonii 18S ribosomal RNA gene), UNCI1RRG Uncinocarpus reesii
18S ribosomal RNA small nucler subunit gene, AOSRR18S Ascosphaeria ap
is 18S ribosomal RNA gene), BYSRR18S Byssochlamys nivea 18S ribosomal RNA
gene), Chaetmium eratum (CEMRR18S Chaetom)
ium elatum 18S ribosomal RNA gene, ERERR18S Eremascus albus 18S ribosomal
RNA gene), Hipomyces cherisospermas (HYY)
RRNA Hypomyces chrysospermus partial 18S ribosomal
RNA sequence), Low Costma Personi (LEARR18)
S Leucostoma persoonii 18S ribosomal RNA, Monascus purpureus18S
ribosomal RNA), Opiostoma urumi (OPHRR18S O
phiostoma ulmi 18Sribosomal RNA)
Stenoceras (OPHRR18SA Ophiostoma stenoceras 18S
ribosomal RNA), Sporotrics schenkyi (OPH)
RR18SB Sporothrix schenckii 18S ribosomal RNA),
Talaromyces flavs (TALRR18STalaromyces flav)
us 18S ribosomal RNA), THSRR18S Thermoascus crustaceus 18S ribosoma
l RNA), tuber melanosporum (TUQRROA Tuber)
melanosporum small subunit ribosomal RNA), rhodosporium dacryiodam (RHDSSRD1 Rhodosporid)
ium dacryoidum gene for small subunit ribosomal DN
A), Symposiomycopsis paphiopedily (SPA
SSRD3 Sympodiomycopsis paphiopedili gene for small
subunit ribosomal DNA).

[Example 2] Detection by agarose gel electrophoresis (1) Preparation of genomic DNA sample Genome DN of the strain used as a PCR sample (cells which had been statically cultured at 25 ° C for 10 days in a potato dextrose liquid medium)
A was prepared according to the CTAB method of Murry et al.
l., Nucleic Acids Res., 8: 4321-4325 (1980)). The strain was used in potato dextrose liquid medium 10
1 g of the cells obtained by static culture at 0 ml for 3 weeks at 25 ° C. is frozen in liquid nitrogen, then crushed in a mortar until it becomes a powder, and 2 × CTAB solution (2% CTAB, 0.1 M Tris-HCl, pH 8.0, 1.
Add 3 ml of 4M NaCl, 1% polyvinylpyrrolidone) and add 55 ml
For 10 minutes. After heating, extraction was performed with 3 ml of a chloroform-isoamyl alcohol (1: 1) solution, and the supernatant was further extracted with 3 ml of a chloroform-isoamyl alcohol (24: 1) solution.
Extracted three times repeatedly. Next, a CTAB solution (10% CTBA, 0.
1/10 volume of precipitation buffer (1% CTAB, 5%
mM Tris-HCl, pH 8.0, 10 mM EDTA) solution was added and mixed, followed by standing at room temperature for 30 minutes. The resulting precipitate is twice precipitated with alcohol, and then treated with a conventional phenol-chloroform (1: 1) solution, and then subjected to ethanol precipitation to perform genome DN.
A sample solution was prepared.

(2) PCR reaction All primers used were those synthesized at Takara Shuzo Co., Ltd. Custom Service Center.

For the PCR reaction, a PCR kit "AmpliTaq DNA polymerase" manufactured by PerkinElmer was used. The reaction was performed using a primer set 5′-GCATCAATACCCAACTGCTTGTC-3 ′ (SEQ ID NO: 9 / comprising SEQ ID NO: 1 / see FIG. 1) and 5′-GACT prepared from the characteristic nucleotide sequence of 18S rDNA of Picium sp.
2 μl of 50 pM of CGAAGAGCCAGAGC-3 ′ (SEQ ID NO: 10 / containing a sequence complementary to SEQ ID NO: 5 / see FIG. 3),
2 μl of various template DNA samples (100 μg / ml), 10 × PCR buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ ,
0.01% gelatin) in 10 μl, 10 mM dATP, dCTP, dGTP,
And dTTP, 2 μl each, sterile distilled water 75.5 μl, `` A
0.5 μl of “mplTaq DNA polymerase” was mixed. P
CR is a DNA thermal cycler (Perkin-Elmer)
The reaction was performed under the conditions of denaturation at 95 ° C for 1 minute, annealing at 60 ° C for 1 minute, extension at 72 ° C for 5 minutes for 25 cycles, and 72 ° C for 7 minutes for 1 cycle.

(3) Detection 3 μl of the reaction solution and a dye solution (0.25% BPB, 0.25% XC, 30%
Glycerol and 1 μl of 10 mM Tris-HCl (pH 8.0) were mixed and electrophoresed on a 2% agarose gel. After staining with ethidium bromide, the DNA band was observed by irradiation with ultraviolet light, and the length of the detected DNA fragment was calculated from the comparison with the migration distance of the molecular weight marker migrated simultaneously with the reaction solution.

As a result, only the DNA fragment corresponding to the theoretical value of about 320 bp appeared in all the samples of the genus Picium.
10A / lane 1: Pythium sylvaticum (Pythium sy
lvaticum) Lane 2: Pythium Graminicora (Pythiu)
m graminicola) Lane 3: Pisium iregurare (Py
thium irregulare) Lane 4: Pythium ultimum Lane 5: Pythium paddicum Lane 6: Pythium vanterpolii Lane 7: Pythium
Aristosporum (Pythium aristosprum) Lane 8:
Pythium torulosum lane
9: Pythium spinosum lane
10: Pythium myriotylu
m)), no DNA fragment was observed in the samples of other genera, Fusarium and Rhizoctonia sp. (FIG. 10B / lane 1: Picium spinosam OPA-1 (Pyt
hium spinosum OPA-1) Lane 2: Pythium myriotylum KPMS Lane 3: Fusarium oxysporum F. SP. Fragariae (Fusarium oxysporum f.sp. fragariae) lane
4: Fusarium solani f. Sp. Phaseoli Lane 5: Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani AG)
-1 Cs-Gi) Lane 6: Rhizoctonia solani AG-2-2
OPR-4 (Rhizoctonia solani AG-2-2 OPR-4)). Also,
In addition, the same detection method was attempted for 18 other genera, but as with Fusarium and Rhizoctonia, about 320 bp of P
The CR product was not amplified. The result is shown in FIG.
1: Mucor sp. Lane 2: Rhizopus oligosporas lane
3: Rhizopus oryzae Lane 4: Nectria glioclad
ioides) Lane 5: Anixiella Reticulata (Anixi)
ella reticulata) and FIG. 11B (lane 1: Pythium spinosum) lane 2: Pythium myriotylum lane 3: Phoma medicaginis lane
4: Penicillium jantinerum
thinellum) Lane 5: Penicillium restrictum Lane 6: Aspergillus niger Lane 7: Aspergillus wentii Lane 8: Trichoderma viride lane
9: Trichodermahamatum Lane 10: Verticillium d
ahliae) Lane 11: Verticillium fungicola var. fungic
ola) Lane 12: Alternaria
ia alternata) Lane 13: Altanaria Altanata
AM20 (Alternaria alternataAM20) lane 14: Acremonium philaophora lane 15: Acremonium sp (Actremonium s)
p.)). As a molecular weight marker, FIG.
A: φX174 / Hinf I digest, Fig. 10B: λ / HindIII
・ EcoRI digest, φX174 / HaeIII digest in Fig. 11A,
In FIG. 11B, λ / HindIII digest was used.

Thus, it was demonstrated that the use of the nucleic acid sequence of the present invention for PCR enables the specific detection of Picium bacteria.

Example 3 Detection by Dot Hybridization with a Labeled Probe (1) Preparation of Biotin-Labeled Probe Within the 18S rDNA amplification range of the genus Pycium amplified with the primer set of (2) of Example 2, the genus Pycium 5'-AGTCTTCGGGCTGGTATTGTGTTGAGTCATAATAA which is a nucleotide sequence characteristic of
CTGTGCGGATCGCACTTTGTGCGATAAATCG-3 '(SEQ ID NO: 11 /
5′-terminal biotin labeled with SEQ ID NO: 2 and 3 / see FIGS. 1 and 2) at Takara Shuzo Co., Ltd. customer service center was used as a labeled probe.

(2) Dot blot hybridization 3 μl of the PCR reaction solution obtained by the operations (1) and (2) of
Denature by heating at 5 ℃ for 10 minutes, spot on nylon membrane (Amersham Hybond-N ⁺ ), irradiate UV for 5 minutes
Was fixed to the membrane. Put this membrane in a hybridization pack and mix hybridization buffer (5x Denhardt's solution, 5x SSC, 0.1% SDS, 0.1% denatured salmon testis DNA)
And the above labeled probe were added, and hybridization was performed at 50 ° C. for 2 hours.

Next, the membrane was treated with a solution containing 2 × SSC and 0.1% SDS at room temperature for 10 minutes and with a solution containing 0.2 × SSC and 0.1% SDS at 55 ° C. for 15 minutes.
For 2 minutes each. Transfer the membrane to a new hybridization pack, react with biotinylated alkaline phosphatase solution at 37 ° C for 30 minutes, and add a chromogenic substrate solution (0.03% nitro blue tetrazolium, 0.03% bromochloroindolyl phosphate) at 37 ° C. The reaction was performed for 1 hour.

(3) Results When the presence or absence of a blue-violet spot was visually determined, the emergence of a blue-violet spot was confirmed in the Picus genus strain, and the sample was determined to be positive. On the other hand, fungi of other genera showed negative, in which no spot appeared.

[0035]

Industrial Applicability According to the present invention, it has become possible to easily and quickly and accurately detect or identify a pathogenic bacterium of the genus Picium which causes a soil disease of a crop from a diseased crop at the genetic level. According to the detection method of the present invention, the causative pathogenic bacteria can be quickly identified in a short time without culturing cumbersome bacteria requiring a long period of time. Therefore, it is possible to take quick control measures without losing time, and furthermore, it is possible to prevent continuous cropping failure by diagnosing the soil before planting, so that the method of the present invention greatly contributes to the agricultural field. There is expected.

[0036]

[Sequence list] SEQ ID NO: 1 Sequence length: 8 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence TGCTTGTC 8 SEQ ID NO: 2 Sequence length Length: 6 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTGCGG 6 SEQ ID NO: 3 Sequence length: 7 Sequence type: Number of nucleic acid strand : Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TAAATCG 7 SEQ ID NO: 4 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear sequence Type: Other nucleic acid Synthetic DNA sequence ATCGATTGAG 10 SEQ ID NO: 5 Sequence length: 5 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCTCT 5 sequence Number: 6 Sequence length: 10 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid DNA sequence GCACACCAGG 10 SEQ ID NO: 7 Sequence length: 12 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence TCATATTTCA GC 12 SEQ ID NO: 8 Sequence Length: 9 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TGAGCTTAG 9 SEQ ID NO: 9 Sequence length: 23 Sequence type: Nucleic acid Number: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GCATCAATAC CCAACTGCTT GTC 23 SEQ ID NO: 10 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Type Sequence type: Other nucleic acid Synthetic DNA sequence GACTCGAAGA GCCAGAGC 18 SEQ ID NO: 11 Sequence length: 66 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence AGTCTTCGGG CTGGTATTGT GTTGAGTCAT AATAACTGTG CGGATCGCAC TTTGTGCGAT 60 AAATCG 66 SEQ ID NO: 12 Length: 1840 SEQ types: the number of nucleic acid strands: single strand Topology: linear sequence type: Genomic DNA Origin Organism: Pythium Miriotirumu (Pythium myriotyl
um) strain name: KPMS sequence CTGGTTGATG CCTGCCAGTA GTCATACGCT TGTCTCAAAG ATTAAGCCAT GCATGTCTAA 60 GTATAAACAA CTTTGTNCTG TGANACTGCG NNTGGCTCAT TATATCAGTT ATAGTCTACT 120 CGATAGTACC TTACTACTTG GATAACCGTA GTAATTCTAG AGCTAATACA TGCATCAATA 180 CCCAACTGCT TGTCGGACGG GTAGCATTTA TTAGATTGAA ACCAATGCAG TCTTCGGGCT 240 GGTATTGTGT TGAGTCATAA TAACTGTGCG GACCGCACTT TTGTGCGGTA AATCGATTGA 300 GTTTCTGCCC TATCAGCTTT GGATGGTAGG ATATGGGCCT ACCATGGCAT TAACGGGTAA 360 CGGGGAATTA GGGNTTGATT CCGGAGAGGG AGCCTTAGAA ACGGCTACCA CATCCAAGGA 420 AGGCAGCAGG CGCGTAAATT ACCCAATCCT GACACAGGGA GGTTAGTGAC AATAAATAAC 480 AATGCTCTGG CTCTTCGAGT CGGGCAATTT GGAATGAGAA CAATTTAAAT CCCTTATCGA 540 GGATCAATTG GAGGGCAAGT CTGGTGCCAG CAGCCGCGGT AATTCCAGCT CCAATAGCGT 600 ATATTAAAGT TGTTGCAGTT AAAAAGCTCG TAGTTGGATT TCTATTTTGA GCGTCCGGTC 660 CGCTCCTTTG GAGTGTGTTA CTAGGATGTT TGAGACATTT TTTGTGAGGA TGTTCTTCTG 720 CCATTCAGTT GGTGGTTGAA TAGACTTGCA TCGTTTACTG TGAAAAAATT AGAGTGTTTA 780 AAGCAGGCGT TTGCTCATTG AATACATTAG CATGGAATAA TAAGATACGA CCTTGGTGG T 840 CTATNTTGTT GGTTTGCACA CCAGGGTCAA TGATTGAATA GGGACAGTTG GGGGTATTCA 900 TATTTCAGCG TCAGAGGTGA AAATTCTTGG ATCGCTGACA AGATGAGCTT AGGCGAAAGC 960 ATTTACCAAG GATGTTTTCA TTAATCAAGA ACGAAAGTTA GGGGATCGAA GATGATTAGG 1020 ATACCCATCG TAGTCCNCAT AAACTATGCC CGATCTCGGG ATTGGCAGTC GTATTTCAAC 1080 ATGCACCTTG CTCAGTCACC CGTATGAGAA AATCAAAGTC TTTGGGTTCC GGGGGGAGTA 1140 TGGTCGCAAG GCTGAAACTT AAAGGAATTG ACGGAAGGGG CACCACCAGG AGTGGAGCCT 1200 GCGGCTTAAT TTGACTCAAC ACGGGAAAAC TTACCAGGTC CAGACATAGT CAAGGATTGA 1260 CAGATTGAGA GCTCTTTCTT GATTCTATGG GTGGTGGTGC ATGNCCGTTC TTAGTTGGTT 1320 GGAGTGATTT GNTCTGGTTA ATTTCCGTTA ACGAACGAGA CCTCCGCGTG CTAAATAGTT 1380 CCGCTTGACA ATTTTTTGTA GGTAGTGGAC TTCTTAGAGG GACTTTTGGG TAATCAAACC 1440 AAAGGAAGTT GGAAGGCAAA TTAACAGGTC TGNTGATGCC CTTAGATGTT CTGGGCCGCA 1500 CGCGCGCTGA CACTGATGCG TTCAACGAGT ATACAACCTT GATCGATAGG TCTGGGTAAT 1560 CTTGTGAATG CGCATCGTGC TAGGGATAGA TTGTTGCAAA CTTTTCAATC TTGAANCGAG 1620 GAATTCCTAG TAACACGCAA GTCATCAGCT TGCATTGATT ACGTCCCTGC CCTTTGTACA 1680 CA CCGCCCGT CGCACCTACC GATTGAATGA CTCGGTGAAG AAATCGGGGA CCGTGAATCT 1740 GNTTGCTTCA TTGCGAGTGG ATTTATGGGG AACTTTTTCT AACCTCGCCA TTTAGAGGAA 1800 GGTGAAGTCG TAACAAGGTT TCCGTAGGTG AACCTGCAGA 1840 Genomic DNA source organism name: Pythium Supinosamu (Pythium spinosum) strain name: OPA-1 sequence CTGGTTGATC CTGCCAGTAG TCATACGCTT GTCTCAAAGA TTAAGCCATG CATGTCTAAG 60 TATAAACAAT TTTGTACTGT GAAACTGCGA ATGGCTCATT ATATCAGNTA TAGTCTACTC 120 GATAGTACCT TACTACTTGG ATAACCGTAG TAATTCTAGA GCTAATACAT GCATANATAC 180 CCAACTGCTT GTCGGGCGGG TAGCATTTAT TAGATTGAAA CCANTGCAGT CTTCGGGCTG 240 GTATTGTGTT GAGTCATAAT AACTGTGCGG ATCGCACTTT GTGCGATAAA TCGATTGAGT 300 TTCTGCCCTA TCAGCTTTGG ATGGTAGGAT ATGGGCCTAC CATGGCATTT AACGGGTTAA 360 CGGGGAATTA GGGTTTTGAT TCCGGAGAGG GAGCCTTAGA AACGGCTACC ACATCCAAGG 420 AAGGCAGGCAGATGATCAGGCAGATGAGCAGGCAGATGAGCAGGCAGATGAAGCGTCAGCAGATGAAGCGTCAGCAGATGCATGAGCACGATCAGGATCAGGAGCTAGAGA TCGAGT CGGGCAATTG GAATGAGAAC AATTTAAATC CCTTAACGAG 540 GATCAATTGG AGGGCAAGTC TGGTGCCACA GCCGCGGTAA TTCCAGCTCC AATAGCGTAT 600 ATTAAAGTTG TTGCAGTTAA AAAGCTCGTA GTTGGATTTC TGTTTTGAGC GTCCGGTCGG 660 CTCCCTCTGG GAGTGTGTAC TTTTGCGACG TTCGAGGCAT TTTTTGTGAG GACGTGTTTC 720 TGCCATTAAG TTGGTGGTTT TGCGGACTTG CATCGTTTAC TGTGAAAAAA TTAGAGTGTT 780 TAAAGCAGGC GTTTGCTCAT TGAATACATT AGCATGGAAT AATAAGATAC GACCTTGGTG 840 GTCTATTTTG TTGGTTTGCA CACCAGGGTA ATGATTAATA GGGACAGTTG GGGGTATTCA 900 TATTTCAGCG TCAGAGGTGA AATTCTTGGA TCNCTGANAG ATGAGCTTAG GCGNAAGCNT 960 TTACCAAGGA TGTTTTCATT AATCAAGAAM GAAAGTNAGG GGATCGAAGA TGATNAGATA 1020 CCCATCGNNN NNTCTNNNCC ATANNCTATG CCGACTCGGG ATTGGCAGTC GNTTNNTTAA 1080 ATGACCTTGT CAGCACCGNA TGAGAGATCC AAAGTCTTTG GGTTCCGGGG GGAGTATGNN 1140 NNAANNCTGA AACTTAAAGG AATTGACGGA AGGGCACCAC CAGGAGTGGA GCCTGCGGCT 1200 TAATTTGACT CAACACGGGG AANNNTNNCA GGTCCAAGAC ATAGTAAGGA TTGACAAGAT 1260 TGAGAGCTCT TTCTTGATTC TATGGGGTGG TGGTGCATGN CCGTTCTNAG TTGGTGGAGT 1320 GATTTGTCTG GTTAATTCCG TTAACGA ACG AGACCTCCGC GTGCTAACTA GTTCCGCTTA 1380 CAATTTTTTG TAGGTTTTGG GACTTCTTAG AGGGACTTTT GGGTAATCAA ACCAAAGGAA 1440 GTTGGAGGCA ATAACAGGTC TGTGATGCCC TTAGNTGTTC TGGGCCGCAC GCGCGCTACA 1500 CTGATGCGTT CAACGAGTAT ACAACCTTGA TCGATAGGTC TGGGTAAGCT TTTGAATGCG 1560 CATCGNNCTA GGGATAGATT GTTGNAATTT TCAATCTTGA ACGAGGAATT CCTAGTAAAC 1620 GCAAGTCATC AGNTTGCATT GATTACGTNC CTGCCCTNTN TACACACCGC CCGTCGCACN 1680 NACCGATTGA ATGACTCGGT GAAAAATTGG GACTGTGAGC CTGTTTGCTT TATTGCGAGT 1740 GGGTTTGTGG GAACTTTTTT TAACCTCGCC ATTTAGAGGA AGGTGAAGTC GTAACAAGGT 1800 TTCCGTAGGT GAACCTGCAG A 1821 SEQ ID NO: 14 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence CTGGTTGATY CTGCCAGT 18 SEQ ID NO: : 15 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GAAACTGCGA ATGGCTCATT 20 SEQ ID NO: 16 Sequence length: 18 Sequence Type: nucleic acid chain Number: single-stranded Topology: linear Sequence type: other nucleic acid Synthetic DNA sequence AGGGYTCGAY YCCGGAGA 18 SEQ ID NO: 17 Sequence length: 19 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: Other nucleic acid Synthetic DNA sequence CGCGGTAATT CCAGCTCCA 19 SEQ ID NO: 18 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TTRATCAAGA ACGAAAGT 18 SEQ ID NO: 19 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence AATTTGACTC AACACGGG 18 SEQ ID NO: 20 Sequence length Length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATAACAGGTC TGTGATGCCC 20 SEQ ID NO: 21 Sequence length: 20 Sequence type: Nucleic acid Strand Number: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA Sequence TTTGYACACA CCGCCCGTCG 20 SEQ ID NO: 22 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence AATGAGCCAT TCGCAGTTTC 20 SEQ ID NO: 23 Sequence Length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence TCTCCGGRRT CGARCCT 17 SEQ ID NO: 24 Sequence length: 18 Sequence type: Nucleic acid chain Number: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence ATTACCGCGG CTGCTGGC 18 SEQ ID NO: 25 Sequence length: 17 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Type Sequence type: Other nucleic acid Synthetic DNA sequence TTGGYRAATG CTTTCGC 17 SEQ ID NO: 26 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CCGTCAATTY YTTTRAGTTT 20 SEQ ID NO: 27 Sequence length: 20 Sequence type: Nucleus Number of acid chains: single-stranded Topology: linear Sequence type: other nucleic acids Synthetic DNA sequence GGGCATCACA GACCTGTTAT 20 SEQ ID NO: 28 Sequence length: 16 Sequence type: Nucleic acid Number of strands: single-stranded Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence GACGGGCGGT GTGTRC 16 SEQ ID NO: 29 Sequence length: 18 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA sequence CYGCAGGTTC ACCTACRG 18

[Brief description of the drawings]

Figure 1: Pythium spinosum OPA-1 (Pythium spinos)
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4). The positions of SEQ ID NO: 1, SEQ ID NO: 9, and SEQ ID NO: 11 (part) are indicated.

Figure 2: Pythium spinosum OPA-1 (Pythium spinos
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4). The positions of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 11 (part) are indicated.

Fig. 3 Pythium spinosum OPA-1 (Pythium spinos)
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4). SEQ ID NO: 5, and SEQ ID NO: 1
The position of the sequence complementary to 0 is indicated.

Figure 4: Pythium spinosum OPA-1 (Pythium spinos
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4). The positions of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 are indicated.

Figure 5: Pythium spinosum OPA-1 (Pythium spinos
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4).

FIG. 6: Pythium spinosum OPA-1 (Pythium spinosum)
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4).

Fig. 7: Pythium spinosum OPA-1 (Pythium spinos
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4).

FIG. 8: Pythium spinosum OPA-1 (Pythium spinosum)
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4).

FIG. 9: Pythium spinosum OPA-1 (Pythium spinosum)
um OPA-1), Psium Milliotirum KPMS (Pythium
myriotylum KPMS), Fusarium oxysporum f.sp.
fragariae), Fusarium solanie F. SP.
Phaseoli, Fusarium solani f.sp.phaseoli, Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani)
AG-1 Cs-Gi), Rhizoctonia solani AG-2-2 OPR-4
It is a figure which shows the base sequence of 18S rDNA of (Rhizoctonia solani AG-2-2 OPR-4).

FIG. 10 is an electrophoresis photograph of 18S rDNA of various fungi PCR-amplified using the primers of the present invention. Lane of FIG. 10A
1 is Pythium sylvaticu
m), Lane 2 is Pythium gramina
inicola), lane 3 is Pythium
irregulare), lane 4 is Pycium Ultimate (Pyt
hium ultimum), lane 5 is Pytium Padicam (Pyt
hium paddicum, lane 6 is Pythium vanterpolii, lane 7 is Pythium aristosprum, lane 8 is Pythium torulosum, and lane 9 is pythium spinosum. ), Lane 10 is Pythium myriotylum, figure
Lane 1 of 10B is the Pysium Spinosum OPA-1 (Pythium
spinosum OPA-1), lane 2 is Pythium myriotylum KPMS, lane 3 is Fusarium oxysporum F. SP. Fragaliae (Fusarium oxysporumf.sp. fragariae), lane 4:
Fusarium Solany F.SP.Faseoli (F
usolium solani f.sp. phaseoli), lane 5 is Rhizoctonia solani AG-1 Cs-Gi (Rhizoctonia solani AG-1)
Cs-Gi), Lane 6 is Rhizoctonia Solany AG-2-2 OP
The amplified DNA of R-4 (Rhizoctonia solani AG-2-2 OPR-4) was electrophoresed.

FIG. 11 is a photograph of electrophoresis of 18S rDNA of various fungi amplified by PCR using the primers of the present invention. Lane of FIG. 11A
1 is Mucor sp., Lane 2 is Rhizopus oligosporas, Lane 3 is Rhizopus oryzae, Lane 4
Is Nectria gliocladioides
cladioides), lane 5 is Anixiella reticulata, lane 1 in FIG. 11B is Pythium spinosum, lane 2 is Pythium myriotylum, lane
3 is Phoma medicaginis, lane 4 is Penicillium
janthinellum), lane 5 is Penicillium restrictum, lane 6 is Aspergillus niger, lane 7 is Aspergillus wentii, lane 8 is Trichoderma virid
e), Trichoderma hamatum in lane 9
hamatum), lane 10 is Verticillium dariae (V
erticillium dahliae, lane 11 is Verticillium fungicola baa.
fungicola), Lane 12 is Alternaria alternata, Lane 13 is Alternaria alternata AM20
0), Lane 14 is Acremonium Philaphora (Acr
emonium philaophora), lane 15 is acremonium
The amplified DNA of SP (Actremonium sp.) Was run.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1: 645) (C12N 1/14 C12R 1: 645) (C12N 15/09 ZNA C12R 1: 645) (72) Inventor Takahashi 241 Kariwano, Nishi-Senhoku-cho, Senboku-gun, Akita Pref.

Claims (4)

[Claims] 1. It comprises any one of the nucleotide sequences of SEQ ID NO: 1 to SEQ ID NO: 8, or a nucleotide sequence complementary thereto.
Oligonucleotides for detecting or identifying fungi belonging to the genus Picium. 2. A probe comprising the oligonucleotide according to claim 1. 3. A primer comprising the oligonucleotide according to claim 1. 4. A method for detecting or identifying a fungus belonging to the genus Picium, comprising using the probe according to claim 2 or the primer according to claim 3.