JP2001314199A - Method for detecting ganoderma boninense which is pathogenic fungus of elaeis guineensis and primer for detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for specifically detecting Ganoderma boninense which is the pathogenic fungus of Elaeis guineensis. SOLUTION: This method for identifying or detecting the Ganoderma boninense, comprising performing a PCR reaction using the chromosomal DNA of a test target microorganism as a template in the presence of a primer corresponding to the base sequence of a region specific to the Ganoderma boninense among the base sequence of the ITS region of a Ganoderma boninense rDNA gene group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an oil palm (El).
Ribosomal DNA of Ganoderma boninense, a common disease fungus of aeis guineensis
(RDNA) intervening gene (Internal transcribed)
Spacer; ITS-1, -2) and Ganoderma using it
Lies in the selective and specific detection of Boninense.

[0002]

BACKGROUND OF THE INVENTION Basal Stem Rot of oil palm caused by the basidiomycete Ganoderma boninense is an important economic fungus in Southeast Asia, especially Malaysia and Indonesia. Stem blight caused by this fungus is 10 years old
It occurred in oil palms for ~ 15 years, and more than 85% of oil palms over 25 years have been infected. It is known that the incubation period of the disease bacteria is several years, and it is known that signs and signs of the disease appear at the end of infection, and more than half of the stem tissues of the late oil palm have become decayed and can no longer be cured. is there.

Since oil palm is a woody plant, it is attracting attention as a source of edible or industrial oils and fats due to its long life and high productivity.
It has been reported to reach over 40%. One of the limiting factors controlling disease is the lack of a diagnostic method that accurately detects the early signs of the disease.

[0004] Ganoderma spp. Which cause diseases (stalk blight) of oil palm as described above are known from Japan,
It is widely distributed in subtropical to tropical regions such as Asia such as Malaysia, Indonesia and Thailand, Africa, Latin America and Colombia. Ganoderma stem blight was first reported in West Africa, and the fungus was named Ganodema lucidum (Wakefield,
EM, 1920, Diseases of the oil palm in West Afric
a. Kew Bulletin 306-308). After that, Ganoderma zonatum (G. zonatum), Ganoderma colossus (G. c.
olossus), Ganoderma escidam (G. encidum), and Ganoderma apranatam (G. applanatum) have been reported (NIFOR, 1978, Nigerian Institute for Oil).
Palm Research, Fourteen Annual Report).

[0005] The disease germs in Malaysia were initially G. luc
Only idum was reported (Thompson, 1931, Stem-rot of t
he oil palm in Malaya, Bulletin, Department of Ag
riculture, Straits Settlements & FMS Science Se
r.6: 23pp). Ganoderma Boninense, G. miniatocinctum (G. miniatocinctum), Ganoderma Charceum (G. chalceum), Ganoderma
G. tornatum, G. zonatum, and G. xylonoides.
xylonoides) were reported one after another (Steyaert, RL, 1
967, Les Ganoderma palmicoles. Bulletin Jardin Bot
anique Nationale Belgique 37: 465-492).

[0006] On the other hand, there is a view that the disease fungi on the Malay Peninsula should be integrated into a kind of Ganoderma boninense (Ho & Nawawi, 1985, Ganoderma boninense).
Pat. From basal stem rot of oil palm in Peninsula
Malaysia. Pertanika 8: 425-428). As described above, several species of the genus Ganoderma have been reported as pathogenic fungi of oil palm, but there has been no experimental demonstration of their pathogenicity. More recently, a series of infection experiments using strains of Ganoderma boninense isolated from oil palm have led to the validation of the pathogenicity of Ganoderma boninense on oil palm (Khairudin, 1990, M.
Sc Thesis. Universiti Pertanian Malaysia, Serdang,
Malaysia and Sariah et al., 1994, Plant Pathology, 43: 5.
07-510).

As a method for separating and culturing Ganoderma boninense, a selective medium (Proceedings of Ganoderma Workshop,
organised by Palm oil Research Institute of Malay
sia, p113-125, 1990). By introducing this medium, it is possible to isolate the mycelium of Ganoderma from infected stems, roots, or soil, but it is difficult to induce fruiting body formation on this artificial medium. The species of the body cannot be identified.

In addition, a recently developed method for detecting Ganoderma boninense uses Enzyme-l using a polyclonal antibody.
There is an inked Immunosorbent Assay (ELISA) (TWD
armono & Suharyanto, Recognition of field material
s of Ganoderma sp.associated with basal stem rot
in oil palm by polyclonal antibody, Menara Perkebu
nan 63 (1): 15-22, 1995). The polyclonal antibody used in this ELISA method does not show cross-reactivity with oil palm tissue cells, but shows weak cross-reactivity with other fungi, so a method for detecting Ganoderma boninense with specific and high sensitivity There is a problem.

[0009]

The gene sequence of an intervening gene (ITS) region of a ribosomal DNA gene group having a sequence specific to Ganoderma boninense, which is a disease of oil palm, is determined. If a sequence can be found, amplifying and detecting the same specific sequence by the PCR method enables highly sensitive and specific detection of diseased bacteria compared to the conventional method.

[0010] It is an object of the present invention to provide a method for finding a specific sequence of the rDNA gene group of Ganoderma boninense, which is a disease of oil palm, and specifically detecting the disease.

[0011]

Ganoderma boninense, which is a disease of oil palm, is distinguished from related bacteria by minute morphological differences such as grain support, basidiomycetes, and basidiospores. Since the morphological characteristics of Ganoderma boninense tend to vary depending on the time of occurrence, environment, and the like, it is very difficult to identify the bacterial species. Under such circumstances, the present inventors considered that comparison at the molecular level is necessary and important for objective classification and identification of Ganoderma boninense.

Therefore, the present inventors determined the nucleotide sequence of the ITS region of the rDNA gene group from the strain belonging to Ganoderma boninense, examined the systematic positional relationship with the related bacteria group,
As a result of intensive research on the detection of a gene sequence specific to Ganoderma boninense, a specific sequence was found,
The inventors succeeded in designing primers capable of specifically detecting those sequences, and have completed the present invention. That is, the present invention is as follows.

(1) A method for identifying or detecting Ganoderma boninense, which comprises detecting a sequence specific to Ganoderma boninense in an intervening gene of ribosomal DNA. (2) The intervening gene of the ribosome DNA is 18S
intervening gene (ITS-1) existing between rDNA and 5.8S rDNA or intervening gene (ITS-1) existing between 5.8S rDNA and 28S rDNA
-2) or the method of (1), which is both of them. (3) Detection of a sequence specific to Ganoderma boninense in the intervening gene of the ribosome DNA was performed by using
The method according to (1) or (2), wherein the amplification is performed by using two types of primers suitable for specific amplification by the R method and using chromosomal DNA or a partial fragment of Ganoderma boninense as a template. the method of. (4) Ganoderma in the intervening gene of ribosomal DNA
The method according to any one of (1) to (3), wherein the sequence specific to Boninense is the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 4 or a part thereof. (5) At least one of the primers has SEQ ID NO: 1.
Or 2 corresponds to the region represented by base numbers 12 to 220, SEQ ID NO: 3 to base numbers 12 to 216, SEQ ID NO: 1 or 2 to base numbers 378 to 573, or SEQ ID NO: 3 to the region represented by base numbers 374 to 569. Any of (1) to (4). (6) One of the primers corresponds to a first region represented by base numbers 12 to 220 in SEQ ID NO: 1 or 2, or base numbers 12 to 216 in SEQ ID NO: 3, and the other of the primers has a sequence Corresponding to the second region represented by base numbers 378 to 573 in No. 1 or 2, or base numbers 374 to 569 in SEQ ID NO: 3, and the first region and the second region (5) The method according to (5), wherein the sequence between the two is amplified. (7) The method according to (5), wherein the other of the primers corresponds to a region in or near the intervening gene. (8) The method according to (6), wherein one of the primers has a base sequence shown in SEQ ID NO: 7 and the other of the primers has a base sequence shown in SEQ ID NO: 8.

(9) 18S rDNA and 5.8S rD
An intervening gene (ITS-1) existing between NA and NA, or an intervening gene (ITS-2) existing between 5.8S rDNA and 28S rDNA, or Ganoderma boninense in both genes. A PCR primer consisting of two oligonucleotides used for identifying or detecting Ganoderma boninense by detecting a unique sequence, wherein one of the primers has a base number of 12 to 12 in SEQ ID NO: 1 or 2. 22
0, or the first region represented by base numbers 12 to 216 in SEQ ID NO: 3, and the other of the primers
PCR corresponding to the second region represented by base numbers 378 to 573 in SEQ ID NO: 1 or 2 or base numbers 374 to 569 in SEQ ID NO: 3, and using the primer
A primer which amplifies a sequence between the first region and the second region. (10) Intervening gene (ITS-1) existing between 18S rDNA and 5.8S rDNA, or
Used to identify or detect Ganoderma boninense by detecting the intervening gene (ITS-2) present between 8S and 28S rDNA or a sequence specific for Ganoderma boninense in both of these genes PC consisting of two types of oligonucleotides
R primer, wherein one of the primers has base numbers 12 to 220 in SEQ ID NO: 1 or 2, base numbers 12 to 216 in SEQ ID NO: 3, base numbers 378 to 573 in SEQ ID NO: 1 or 2, or SEQ ID NO: 3. Wherein the other of the primers corresponds to a region in or near the intervening gene. Hereinafter, the present invention will be described in detail.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The method of the present invention is a method for identifying or detecting Ganoderma boninense. Ganoderma
Identification or detection of Boninense means determining the microorganism to be tested as Ganoderma boninense or another microorganism, or determining the presence or absence of Ganoderma boninense in the test subject.

The method of the present invention is carried out by detecting whether or not the intervening gene present in the ribosomal DNA of the microorganism chromosome in the subject has a sequence specific to Ganoderma boninense.

The sequence specific to Ganoderma boninense in the intervening gene of ribosomal DNA includes the sequence included in the intervening gene of ribosome DNA of Ganoderma boninense,
It refers to a sequence that is not included in the intervening gene of ribosomal DNA of other closely related species such as Ganoderma.

In the 1980's, the technical progress of molecular biology,
In particular, cloning technology, PCR (Polymerase chain Reacti
on) method, the development of automatic sequencers, the spread of computers capable of high-speed calculations, and the development of new molecular evolution analysis programs have created molecular phylogenetics, which study biological relationships and phylogenetic evolution from the molecular level. Since molecular evolution changes at a nearly constant rate compared to morphology, this can be regarded as a "molecular clock", and phylogenetic relationships between organisms can be estimated by comparing information macromolecules such as DNA, RNA or proteins of organisms. I came to think. Analysis of the base sequence of ribosomal RNA (rRNA) has been incorporated in fungi, and data is being accumulated.

[0019] Ribosomal RNA is involved in the synthesis of proteins essential for the vital activities of all living organisms, and the gene encoding it, ribosomal DNA (rDNA), exists in multiple copies in the genome of most living organisms, and is eukaryotic. It is known that there are hundreds to thousands of copies in living organisms (Gerbi, S. et al.
A. 1985, Moleculasr Evolutionary Genetics. PlenumP
ress. New York, pp419-517, and Appels, R. & Honeyc
utt, RL, 1986, DNASystematics. CRC Press, Boca
Raton, Florida, pp81-135). In eukaryotes, generally, regions encoding 18S rRNA, 5.8S rRNA and 28S rRNA (referred to as 18S rDNA, 5.8S rDNA and 28S rDNA, respectively) are continuously present in the eukaryote, and the repetitive sequence is repeated. In this case, there are intervening genes (ITS-1, ITS-2) between 18S and 5.8S and between 5.8S and 28S (see FIG. 1).

In a systematic study of a molecular system, it is common to compare genes considered to be homologous or their intervening genes. For example, 18S rDNA is a family or orde
r) or for the elucidation of the phylogenetic relationship of higher taxa above the class (Bowman, BH, et al. Mol. Biol.
Evol. 9: 285-296). Also, 28S rDNA has been used for phylogenetic analysis at the genus and species level. Examples of reported microorganisms include Ganoderma (Ganoderm
a) Genus (Moncalvo, J.-M. et al., 1995, Mycologia, 8
7: 223-238).

On the other hand, an intervening gene (ITS-1, -2), which is richer in the variable region than 18S and 28S, is used as an index for identifying a closely related or intraspecific family group. Examples of basidiomycetes reported include the genus Dermocybe and the genus Cortinarius (Liu, YJ et al., 1997, Ca.
n. J. Bot. 75: 519-532), Armillari
a) Genus (Chillali, M. et al., 1998, New Phytol. 138:
553-561) and Ganoderma lucidam (Ganoderma luci)
dum) complex species (Moncalvo, J. -M., et al., 1995, Mycol.
Res., 99: 1489-1499), Ganoderma Lucidum (Ganod
erma lucidum) complex species (Hseu, R.-S., et al., 1996,
Appl. & Environ. Microbiol., 62: 1354-1363).

Against this background, 1) Based on comparative analysis of intervening genes (ITS-1, ITS-2), which are used as excellent indicators for discriminating closely related or intraspecies strains, Ganoderma boninense, a disease of palm
rma boninense), and 2) Ganoderma boninense based on the obtained molecular information (base sequence of ITS-1, -2).
An attempt was made to analyze a unique sequence specific to Ganoderma boninense. Then, as shown in Examples described later, a sequence specific to Ganoderma boninense was found, and a primer capable of specifically detecting those sequences was successfully designed.

That is, in the present invention, Ganoderma
Ribosomal DNA to detect sequences specific to Boninense
Intermediates of 18S rDNA and 5.8S
an intervening gene (ITS-1) present between rDNA and
Alternatively, an intervening gene (ITS-2) existing between 5.8S rDNA and 28S rDNA or both of them may be mentioned.

The sequence specific to Ganoderma boninense in the intervening gene of ribosomal DNA is detected by using primers suitable for specifically amplifying this sequence by PCR, using the chromosomal DNA or partial fragment of Ganoderma boninense. Can be carried out by an amplification reaction using as a template.

The sequence specific to Ganoderma boninense in the intervening gene of ribosomal DNA includes SEQ ID NO: 1
Or a part thereof. The nucleotide sequences shown in SEQ ID NOs: 1 to 3 are Ganoderma boninense MCI3595 and Ganoderma boninense MCI, respectively.
3597, and two synthetic primers ITS1 and ITS4 (Michael A.I.) designed to amplify genes (including 5.8S rDNA) in the ITS region of Ganoderma boninense MAFF305601.
nnis, David H. Gel et al., PCR Experiment Manual (HBJ Publishing Office), and the base sequence of a DNA fragment amplified by PCR using chromosomal DNA of each strain as a template (see FIG. 1).

FIGS. 4 and 5 show a part of the nucleotide sequence (5.8S rDNA and a part of ITS-1 and ITS-2 present on both sides thereof) and a corresponding closely related ITS. 3 shows a comparison of nucleotide sequences of regions. The primer used in the present invention is not particularly limited as long as it can specifically amplify the ITS region of Ganoderma boninense.
5 and FIG. 5, a region specific to Ganoderma boninense may be set, and an oligonucleotide having a base sequence corresponding to the region may be synthesized. The suitability of the synthesized oligonucleotide is that an amplification product is obtained when the chromosomal DNA or partial fragment of Ganoderma boninense is used as a template, and an amplified product is obtained when the chromosomal DNA or partial fragment of a closely related species is used as a template. You can find out by checking that there are no.

Specific examples of the primer used in the present invention include base numbers 12 to 220 in SEQ ID NO: 1 or 2.
Alternatively, a primer (5′-side primer) corresponding to the first region represented by base numbers 12 to 216 in SEQ ID NO: 3 and base numbers 374 to 5 in SEQ ID NO: 1 or 2
69 or base numbers 378 to 573 in SEQ ID NO: 3
And a primer (3′-side primer) corresponding to the second region represented by When the sequence between the first and second regions is amplified by these primers, IT
D containing S-1 and ITS-2 and 5.8S rDNA sandwiched between them
An NA fragment is obtained.

More specifically, the primers include a 5'-side primer having the nucleotide sequence shown in SEQ ID NO: 7 (base numbers 84 to 107 in SEQ ID NO: 1) and a 3'-side primer having the nucleotide sequence shown in SEQ ID NO: 8 And primers (base numbers 437 to 459 in SEQ ID NO: 1). In addition, a 5′-side primer having the nucleotide sequence shown in SEQ ID NO: 9 (base numbers 98 to 117 in SEQ ID NO: 1) and a 3′-side primer having the nucleotide sequence shown in SEQ ID NO: 10 (base numbers 573 to 573 in SEQ ID NO: 1) 552) can also be used in the present invention. Furthermore, a combination of the 5 'primer shown in SEQ ID NO: 7 and the 3' primer shown in SEQ ID NO: 10, or the combination of the 5 'primer shown in SEQ ID NO: 9 and the 3' primer shown in SEQ ID NO: 8 may be used. In addition, the primer used in the present invention may have at least one corresponding to a sequence specific to Ganoderma boninense in an intervening gene of ribosomal DNA, and the other primer corresponds to a region in or adjacent to the intervening gene. The sequence does not need to be specific to Ganoderma boninense. That is, even if the other primer corresponds to a region common to the genus Ganoderma or a microorganism closely related thereto, if one primer is specific to Ganoderma boninense, the chromosomal DNA or partial fragment of Ganoderma boninense Only in the case where is used as a template, an amplification product is generated by PCR, so that Ganoderma boninense can be identified or detected. For example, ITS-
When a primer specific to Ganoderma boninense corresponding to the upstream region of No. 1 was used, the downstream region of ITS-1 was 5.8 S rDN as the 3 ′ primer.
It may be a primer corresponding to any of A, ITS-2 and 28S rDNA. Alternatively, a primer corresponding to the second region exemplified as the above-mentioned 3 ′ primer may be used as a 5 ′ primer, and a primer corresponding to a downstream region may be used as a 3 ′ primer.

Techniques such as preparation of chromosomal DNA, synthesis of oligonucleotides, PCR reaction and analysis of PCR products are as follows.
Methods well known to those skilled in the art can be employed.

[0030]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

[0031]

Example 1 Phylogenetic Consideration by Analyzing the Nucleotide Sequence of the Intermediate Gene of Ganoderma boninense The nucleotide sequence of the intervening gene and phylogenetic analysis were performed on this strain as follows.

[Test strain] Ganoderma boninense MCI35
95 Ganoderma boninense MCI35
97 Ganoderma boninense MAFF3
05601 Ganoderma lucidum MAFF42000
9

Among the above strains, Ganoderma boninense MCI3
595 and MCI3597 were obtained by collecting the fruiting body of Ganoderma from an oil palm plantation on the island of North Sumatra, Indonesia, and obtaining a culture.Then, the morphological and cultural characteristics were examined and identified. This is the strain for which Ga
noderma boninense MAFF305601 and Ganoderma lucidum
MAFF420009 is a reference strain obtained from the Ministry of Agriculture, Forestry and Fisheries, National Institute for Agrobiological Resources.

[Experimental method] (1) Culture method of strains A malt medium (malt powder 2%, polypeptone 0.1%, glucose 2%) was dispensed in a volume of 40 ml into a 200 ml Erlenmeyer flask, and sterilized in an autoclave. One strain was inoculated and cultured with shaking at 150 rpm at 20 ° C. or 24 ° C. for 7 to 10 days. After the culture, the cells were collected by filtration with a filter (Toyo Filter Paper No. 101).

(2) Extraction of DNA DNA was extracted from the cells by using a DNA extraction reagent (Plant DNAzol Reagent, manufactured by GIBCO BRL) according to the method of use.

(3) Amplification of Intermediate Gene (ITS) Region Gene (including 5.8S rDNA) by PCR The PCR amplification of the ITS region gene (including 5.8S rDNA) is obtained in (2) above. Two types of synthetic primers ITS1 and ITS4 (Michael A.I.) designed to amplify ITS region genes (including 5.8S rDNA) using 100ng of DNA
nnis, David H. Gel et al., PCR Experiment Manual (HBJ Publishing Bureau).

The composition of the reaction solution was 0.2 μM 5 ′ primer (ITS1), 0.2 μM 3 ′ primer (ITS4), 10 mM Tris
-HCl (pH 8.3), 50 mM potassium chloride, 1.25 mM magnesium chloride, 0.2 mM each deoxyribonucleotide (dATG, dGT
P, dCTP, dTTP) and 0.04 U / μl Taq DNA polymerase (TaKaRa Taq manufactured by Takara Shuzo Co., Ltd.) to adjust the total liquid volume to 20 μl. The conditions are as follows: after incubating at 96 ° C for 1 minute,
A cycle of 45 seconds, 55 ° C. for 1 minute and 30 seconds, and 72 ° C. for 2 minutes was performed 25 times, and the final cycle was kept at 72 ° C. for 4 minutes, and PCR (PTC-100 manufactured by MJ Research Inc., use)
Was done. FIG. 1 shows the approximate positions of the primers used in Examples 1 and 2 in the ITS region.

After completion of the PCR, 20 μl of the reaction solution was subjected to 1.8% agarose (Nusive 3: 1 Agarose (manufactured by FMC BioProducts)) gel electrophoresis (100 V, 30 minutes). 0.2 μg / ml after electrophoresis
Stained with ethidium bromide solution for 25 minutes, cut out the amplified target ITS region gene (including 5.8S rDNA),
After the gel was dissolved by incubating at 10 ° C. for 10 minutes, phenol / chloroform extraction and ethanol precipitation were performed to recover DNA fragments.

(4) Cloning The recovered DNA fragment was ligated to an E. coli plasmid vector (Nova) using a ligation kit (Takara Shuzo Co., Ltd.).
gen, pT7 vector), and the obtained recombinant plasmid was used to transform Escherichia coli DH5α.

(5) Determination of Nucleotide Sequence and Phylogenetic Analysis The nucleotide sequence was determined from the transformant using the desired plasmid DN.
A was extracted, and a sequencer (ABI377, manufactured by Applied Biosystems) and a DNA sequencing kit (manufactured by Applied Biosystems, Big Dye Terminater cyc)
le sequencing Ready Reaction) according to the kit. The determined ITS region gene (5.8S
The sequences (including rDNA) are described in the sequence listing as follows.

Ganoderma bonince
nense) MCI3595: SEQ ID NO: 1 Ganoderma boninense MCI35
97: SEQ ID NO: 2 Ganoderma boninense MAFF3
05601: SEQ ID NO: 3 Ganoderma boninense MAFF420
009: SEQ ID NO: 4

Alignment of the nucleotide sequence data was performed using software CLUSTAL W (JD Thompsonst al., 1994, Nucl.
Acid Res. 22: 4673-4680). The phylogenetic tree is
Phylogenetic tree creation package software PHYLIP (J. Felsenstein.
PHYLIP-Phylogenetic inference pacage, version 3.55
C, Computer programs distributed by the auther, Dep
ertment of Genetics, Univ. of Washington, Seattle)
Was created by the Neighbor-joining method (FIGS. 2 and 3).

For reference in phylogenetic analysis, Genbank (N
ITS base sequence data registered in the National Center for Biotechnology Information) was used. The number added after the bacterial name is the bacterial strain number and the accession number of the nucleotide sequence registered in Genbank.

Ganoderma ahmadi
i) FWP14329: Z37047, Z37098 Ganoderma carnosum JAHN11
97-121: Z37057, Z37082 Ganoderma lucidum RSH RZ: X78
743, X78764 Ganoderma lucidum RSH 0626: Z
37048, Z37072 Ganoderma lucidum ATCC 32471:
X78744, X78765 Ganoderma lucidum JMM P93-1:
X78745, X78766 Ganoderma lucidum RYV33217: Z
37096, Z37073 Ganoderma lucidum CBS270.81:
Z37049, Z37099 Ganoderma lucidum HMAS60537:
Z37050, Z37074 Ganoderma lucidum CBS430.84:
Z37051, Z37075 Ganoderma lucidum ATCC46755:
Z37052, Z37076 Ganoderma lucidum RSH TEX.1:
Z37053, Z37077 Ganoderma oregonense CBS1
77.30: Z37060, Z37100 Ganoderma oerstedii ATCC4
6750: Z37061, Z37101 Ganoderma oerstedii ATCC5
2409: Z37058, Z37083 Ganoderma oerstedii ATCC5
2410: X78739, X78760 Ganoderma pfeifferi CBS
747.84: X78738, X78759 Ganoderma resinaceum CBS1
94.76: X78737, X78758 Ganoderma resinaceum CBS1
52.27: Z37062, Z37085 Ganoderma resinaceum ATCC
52411: Z37059, Z37084 Ganoderma tsugae RSH J2: X7874
6, X78767 Ganoderma tsugae RSH 1109: X787
47, X78768 Ganoderma tsugae RSH BLC: Z3709
7, Z37078 Ganoderma tsugae RSH 0981: X787
48, X78769 Ganoderma tsugae CBS223.48: Z37
054, Z37079 Ganoderma tsugae CBS428.84: X78
735, X78756 Ganoderma tsugae ATCC 46754: Z37
055, Z37080 Ganoderma valesiacum CBS2
82.33: Z37056, Z37081 Cantharellus cibarius3
6066: AF044689

(6) Phylogenetic considerations FIG. 2 shows a phylogenetic tree based on the nucleotide sequence of ITS-1. ITS
Ganoderma boninense from Sumatra
(MCI3595 and MCI3597) is G. boninense (MAFF305601)
And the same lineage, and found to be independently located near Ganoderma lucidum. G. boninense (X78749) registered in Genbank suggested that Ganoderma boninense analyzed by the present inventors is located in a different lineage group.

FIG. 3 shows a phylogenetic tree based on the nucleotide sequence of ITS-2. As a result of phylogenetic analysis, Ganoderma bo from Sumatra
ninense (MCI3595 and MCI3597) is G. boninense (MAFF3
05601) and form the same branch as G. oregonense, G. tsu
gae, G. oerstedii, G. lucidum, G. valesiacum, G. a
hmadii and G. carnosum were found to be located independently. In addition, it was suggested that it belongs to a different family from G. boninense (X78749).

G. boninense (X78749) was described in 1990 by R.-
Separated from sawdust in Taiwan by S. Hseu, G. bo
Although it is a strain identified as ninense, the original growth place of G. boninense is limited to oil palm, and it is systematically different from the ITS results of G. boninense analyzed by the present inventors. This strain needs to be reconsidered. These results strongly suggest that the nucleotide sequence of the intervening gene (ITS-1, -2) is effective as an index for identifying closely related or intraspecific families of Ganoderma boninense.

[0048]

Example 2 Detection of Ganoderma boninense By aligning the nucleotide sequence data obtained in Example 1, Ganoderma boninense MCI3595, MCI
A specific sequence common to 3597 and MAFF305601 was found, and the following primers could be designed. Using these primers, the validity of the detection range for Ganoderma boninense and 13 related species thereof and 6 related genera of the genus Ganoderma was confirmed.

[Test strain] 1. Ganoderma boninense MCI3
595 2. Ganoderma boninense MAFF
305601 3. Ganoderma adspersum
CBS351.74 4. Ganoderma applantum CB
S175.30 5. Ganoderma colossum CBS26
7.88 6. Ganoderma lobatum CBS222.4
8 7. Ganoderma lucidum MAFF4200
09 8. Ganoderma meredithae C
BS269.88 9. Ganoderma oerstedii
CBS514.92 10.Ganoderma oregonense C
BS264.88 11.Ganoderma pfeifferi
CBS221.48 12.Ganoderma resinaceum C
BS152.27 13.Ganoderma tornatum IMI34
4769 14.Ganoderma valesiacum C
BS282.33 15.Ganoderma weberianu
m) CBS219.36 16.Amauroderma rude CBS209.49 17.Daedalea querina CBS221.61 18.Fomes fomentarius CB
S311.82 19.Fomitopsis pinicola CB
S221.39 20.Phanerochaete chrysosporium
chrysosporium) CBS481.73 21.Polyporus squamosus
CBS425.48

Among the above strains, Ganoderma boninense MCI3
Strains other than 595 are from the Ministry of Agriculture, Forestry and Fisheries,
AFF), Central Bureauschule Cultures (C
BS), and a strain obtained from the International Mycological Institute (IMI).

[Experimental method] (1) Culture method of strain and DNA extraction method Culture of the strain and extraction of DNA were carried out according to (1) of Example 1,
This was performed according to the method described in (2).

(2) Amplification of Partial Nucleotide Sequence of 18S rDNA Gene by PCR and Confirmation of Amplification The amplification of the 18S rDNA gene was performed using the DNA 50 obtained in the above (1).
Using pg as a template and the following two types of synthetic primers designed on 18S rDNA, the partial base sequence (about 840 b
p) was amplified.

(5′-side primer) 5′-CTACTGCGAAAGCATTGCCAAGG-3 ′ (SEQ ID NO: 5) (3′-side primer) 5′-CCTTGTTACGACTTTTACTTCCTC-3 ′ (SEQ ID NO: 6))

The composition of the reaction solution was the same as that described in Example 1 (3) except that the antibody (CLONTE) of 8.8 ng / μl Taq DNA polymerase was used.
Add Taq Start Antibody (CH Co., Ltd.) to make the total volume 20
It was adjusted to be μl. The conditions were as follows: after incubating at 96 ° C for 1 minute, then perform 40 cycles of 96 ° C for 20 seconds, 50 ° C for 20 seconds, and 72 ° C for 1 minute.
Incubation for 4 minutes in PCR (MJ Research Inc. P
Using TC-100). The DNA was dispensed in small quantities into sterile microtubes immediately before PCR, and
After preserving the temperature, PCR was performed.

After completion of the PCR, 3 μl of the reaction solution was subjected to 1.2% agarose (SEA KEM GTG Agarose ((FMC BioProducts))) gel electrophoresis (100 V, 30 minutes).
Stained with ethidium bromide solution for 25 min.
It was confirmed that the NA gene (about 840 bp) was amplified (FIG. 6-A), and that the DNA obtained in (1) could be used for PCR.

(3) Ganoderm Boninense
a boninense) Design of specific primer Based on the alignment data obtained in Example 1 (5),
A specific sequence possessed only by Ganoderma boninense was found, and the following primers were designed. This primer is shown in FIGS. 3 and 4 as an example of the design of a Ganoderma boninense-specific primer.

(5 'primer) 5'-GCACTTACTGTGGGTTATAGATCG-3' (SEQ ID NO: 7) (3 'primer) 5'-GAGAGGAGCCGATCAATAAAGA-3' (SEQ ID NO: 8)

(4) ITS region gene (5.8S rDN by PCR)
A) Partial nucleotide sequence amplification of ITS region gene (including 5.8S rDNA)
370 bp) was amplified using the 50 pg of DNA obtained in the above (1) as a template and the two synthetic primers (SEQ ID NO: 7 and SEQ ID NO: 8) of the above (3) to detect Ganoderma boninense. Was. PCR was performed by the method of the above (2). PCR conditions were as follows: after incubating at 96 ° C for 1 minute,
The cycle was performed 20 times at 20 ° C., 20 seconds at 64 ° C., and 1 minute at 72 ° C. for 40 times, and the PCR was carried out at a final temperature of 72 ° C. for 4 minutes. After completion of PCR, add 3 μl of the reaction solution to 1.8%
Agarose (Nusive 3: 1 Agarose (manufactured by FMC BioProducts)) gel electrophoresis (100 V, 30 minutes) was used. 0.2 after electrophoresis
Staining was performed with a μg / ml ethidium bromide solution for 25 minutes.

(5) Results It was confirmed that the target ITS region gene (including 5.8S rDNA) was amplified only by Ganoderma boninense (FIG. 6-B). From the above results, it was found that the specific primer obtained in (3) of Example 2 can be used for detecting Ganoderma boninense.

[0060]

Industrial Applicability According to the present invention, Ganoderma boniense, which is a common germ of oil palm, is used.
nense) can be detected specifically and with high sensitivity, which proved to be a very effective method for early diagnosis of diseased bacteria. Ganoderma Boninense (Ganoderm
a boninense), which has a great effect on elucidation of the interaction between diseased bacteria and plant cells, such as elucidation of the infection mechanism, investigation of the source of infection, and tracking of the diffusion pathway.

[0061]

[Sequence List] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation Badan Pengkazian Dan Penelapan Technology tea. Bakri and Brothers Bioindustry Association Director of Industrial Technology Koji Kajimura <120> Detection method of oil palm disease ganoderma boninense and primer for detection <130> J04505 <140> <141> 2000-05-09 <160> 10 <170> PatentIn Ver. 2.0

[0062] <210> 1 <211> 616 <212> DNA <213> Ganoderma boninense <400> 1 aaggatcatt atcgagtttt gactgggttg tagctggcct tccgaggcat cgtgcacgcc 60 ctgctcatcc actctacacc tgtgcactta ctgtgggtta tagatcgtgt ggagcgagct 120 cgttcgtttg acgagttcgc gaagcgcgtc tgtgcctgcg ttttatcaca aacactataa 180 agtattagaa tgtgtattgc gatgtaacgc atctatatac aactttcagc aacggatctc 240 ttggctctcg catcgatgaa gaacgcagcg aaatgcgata agtaatttga attgcagaat 300 tcagtgaatc atcgaatctt tgaacgcacc ttgcgctcct tggtattccg aggagcatgc 360 ctgtttgagt gtcatgaaat cttcaaccta caatctcttt gcggtttttg taggcttgga 420 cttggaggct tgtcggtctt ttattgatcg gctcctctca aatgcattag cttggttcct 480 ttgcgaatcg gctgtcggtg tgataatgtc tacgccgcga ccgtgacgcg tttggcgagc 540 ttctaaccgt cccgttattg ggacaactct tatgacctct gacctcaaat caggtaggac 600 tacccgctga acttaa 616

[0063] <210> 2 <211> 616 <212> DNA <213> Ganoderma boninense <400> 2 aaggatcatt atcgagtttt gactgggttg tagctggcct tccgaggcat cgtgcacgcc 60 ctgctcatcc actctacacc tgtgcactta ctgtgggtta tagatcgtgt ggagcgagct 120 cgttcgtttg acgagttcgc gaagcgcgtc tgtgcctgcg ttttatcaca aacactataa 180 agtattagaa tgtgtattgc gatgtaacgc atctatatac aactttcagc aacggatctc 240 ttggctctcg caccgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat 300 tcagtgaatc atcgaatctt tgaacgcacc ttgcgctcct tggtattccg aggagcatgc 360 ctgtttgagt gtcatgaaat cttcaaccta caatctcttt gcggtttttg taggcttgga 420 cttggaggct tgtcggtctt ttattgatcg gctcctctca aatgcattag cttggttcct 480 ttgcgaatcg gctgtcggtg tgataatgtc tacgccgcga ccgtgacgcg tttggcgagc 540 ttctaaccgt cccgttattg ggacaacgct tatgacctct gacctcaaat caggtaggac 600 tacccgctga acttaa 616

[0064] <210> 3 <211> 612 <212> DNA <213> Ganoderma boninense <400> 3 aaggatcatt atcgagtttt gactgggttg tagctggcct tacgaggcat cgtgcacgcc 60 ctgctcatcc actctacacc tgtgcactta ctgtgggtta tggatcgtgt ggagcgagct 120 cgtttgacga gtttgcgaag cgcgtctgtg cctgcgtttt atcacaaaca ctataaagta 180 ttagaatgtg tattgcgatg taacgcatct atatacaact ttcagcaacg gatctcttgg 240 ctctcgcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg cagaattcag 300 tgaatcatcg aatctttgaa cgcaccttgc gctccttggt attccgagga gcatgcctgt 360 ttgagtgtca tgaaatcttc aacctacaat ctctttgcgg tttttgtagg cttggacttg 420 gaggcttgtc ggtcttttat tgatcggctc ctctcaaatg cattagcttg gttcctttgc 480 gaatcggctg tcggtgtgat aatgtctacg ccgcgaccgt gacgcgtttg gcgagcttct 540 aaccgtcccg ttattgggac aactcttatg acctctgacc tcaaatcagg taggactacc 600 cgctgaactt aa 612

[0065] <210> 4 <211> 597 <212> DNA <213> Ganoderma lucidum <400> 4 aaggatcatt atcgagtttt gaccgggttg tagctggcct tccgaggcat gtgcacgccc 60 tgctcatcca ctctacacct gtgcacttac tgtgggcttc agattgcgag gcacgctctt 120 taccgggctt gcggagcata tctgtgcctg cgtttatcac aaactctata aagtaacaga 180 atgtgtattg cgatgtaaca catctatata caactttcag caacggatct cttggctctc 240 gcatcgatga agaacgcagc gaaatgcgat aagtaatgtg aattgcagaa ttcagtgaat 300 catcgaatct ttgaacgcac cttgcgctcc ttggtattcc gaggagcatg cctgtttgag 360 tgtcatgaaa tcttcaacct acaagctttt gtggtttgta ggcttggact tggaggcttg 420 tcggccgtta tcggtcggct cctcttaaat gcattagctt ggttccttgc ggatcggctc 480 tcggtgtgat aatgtctacg ccgcgaccgt gaagcgtttg gcgagcttct aaccgtctta 540 taagacagct ttatgacctc tgacctcaaa tcaggtagga ctacccgctg aacttaa 597

<210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 5 ctactgcgaa agcatttgcc aagg 24

<210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 6 ccttgttacg acttttactt cctc 24

<210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 7 gcacttactg tgggttatag atcg 24

<210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 8 gagaggagcc gatcaataaa aga 23

<210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 9 ttatagatcg tgtggagcga 20

<210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR <400> 10 ataagagttg tcccaataac gg 22

[Brief description of the drawings]

FIG. 1 is a diagram showing a rough position of a primer used in Examples 1 and 2 with respect to a ribosomal DNA gene group.

FIG. 2 is a diagram showing a phylogenetic relationship of the genus Ganoderma based on the nucleotide sequence of the ITS1 gene.

FIG. 3 is a diagram showing a phylogenetic relationship of the genus Ganoderma based on the nucleotide sequence of the ITS2 gene.

FIG. 4 is a diagram showing a design example of a primer specific to the 5 ′ side of Ganoderma boninense. Gamoderma boninense Designed to be specific for Ganoderma boninense, showing a comparison of the nucleotide sequences of MCI3595 and the ITS1 gene of the genus Ganoderma
An example of the 5 'primer (SEQ ID NO: 7) is shown. "." Indicates MC
"-" Indicates that the base is the same as that of I3595, "-" indicates that the base is the same, and "*" indicates that all the compared sequences are the same base.

FIG. 5 is a diagram showing an example of designing a 3′-side specific primer of Ganoderma boninense. Gamoderma boninense MCI3595
And comparison of the nucleotide sequence of the ITS2 gene of the genus Ganoderma, designed to be Ganoderma boninense-specific
An example of the 3'-side primer (SEQ ID NO: 8) is shown. "." Indicates MC
"-" Indicates that the base is the same as that of I3595, "-" indicates that the base is the same, and "*" indicates that all the compared sequences are the same base.

FIG. 6: Detection of Ganoderma boninense by PCR. A
Shows the result of electrophoresis of the PCR product obtained in Example 2 (2), and B shows the result of electrophoresis of the PCR product obtained in Example 2 (4). “M” indicates the molecular weight marker, and “N” indicates the result when no template DNA was added.
The number of each lane corresponds to the number of the test strain in Example 2.

 ──────────────────────────────────────────────────続 き Continued on the front page (71) Applicant 594201744 Jl. M. H. Thamrin No. 8, Jakarta 10340, Indonesia (71) Applicant 594201755 p. tea. Bakley and Brothers T. Bakrie and Brothers Indonesia, Jakarta 12920, JEL. H. R. Rasuna Sayed Cub. Bee-1 Jl. H. R. Rasuna Said Kav. B-1, Jakart a 12920, Indonesia (71) Applicant 597031070 Bioindustry Association 2-26-9 Hatchobori, Chuo-ku, Tokyo (74) The above four agents 100089244 Patent Attorney Tsutomu Toyama (two other) 71) Applicant 000001144 Director of the Industrial Technology Institute 1-3-1 Kasumigaseki, Chiyoda-ku, Tokyo (74) One of the above-mentioned sub-agents 100089244 Patent Attorney Tsutomu Toyama (two other) (72) Inventor Akiko Sakamoto Yokohama-shi, Kanagawa 1000 Kamoshita-cho, Aoba-ku, Yokohama Research Laboratory, Mitsubishi Chemical Co., Ltd. (72) Inventor Takashi Mikawa 1000 Kamoshita-cho, Aoba-ku, Yokohama, Kanagawa Prefecture, Yokohama Research Laboratory of Mitsubishi Chemical Corporation (72) Inventor Makoto Murase, Yokohama City, Kanagawa 1000 Kamoshita-cho, Aoba-ku Mitsubishi Chemical Corporation Yokohama Research Laboratory (72) Inventor Akira Tanaka 1000 Kamoba-cho, Aoba-ku Yokohama-shi, Kanagawa Prefecture Mitsubishi Chemical Corporation Yokohama Research Laboratory (72) Inventor Ryuichiro Kurane Tsukuba, Ibaraki Prefecture City East 1 chome 3 Industrial Technology Institute Life Institute of Advanced Industrial Science and Technology in the F-term (reference) 4B024 AA07 AA11 CA01 CA20 4B063 QA18 QQ07 QQ42 QQ50 QR08 QR42 QR62 QS16 QS25 QX01

Claims (10)

[Claims] 1. A method for identifying or detecting Ganoderma boninense, comprising detecting a sequence specific to Ganoderma boninense in an intervening gene of ribosomal DNA. 2. The method according to claim 2, wherein the intervening gene of the ribosomal DNA is
Intervening gene (ITS-1) existing between 18S rDNA and 5.8S rDNA, or 5.8S rDNA
The method according to claim 1, which is an intervening gene (ITS-2) present between DNA and 28S rDNA or both. 3. A method for detecting a sequence specific to Ganoderma boninense in the intervening gene of the ribosome DNA, using two types of primers suitable for specifically amplifying the sequence by PCR. 3. The method according to claim 1 or 2, wherein the method is carried out by an amplification reaction using the chromosomal DNA or partial fragment of the above as a template. 4. A sequence specific to Ganoderma boninense in an intervening gene of ribosomal DNA is represented by SEQ ID NOS: 1-4.
2. The nucleotide sequence according to claim 1 or a part thereof.
The method according to any one of claims 1 to 3. 5. At least one of the primers comprises nucleotides 12 to 220 in SEQ ID NO: 1 or 2, nucleotides 12 to 216 in SEQ ID NO: 3, SEQ ID NO: 1 or 2
The method according to any one of claims 1 to 4, which corresponds to a region represented by base numbers 378 to 573 in SEQ ID NO: 3 or 374 to 569 in SEQ ID NO: 3. 6. One of the primers corresponds to a first region represented by base numbers 12 to 220 in SEQ ID NO: 1 or 2, or base numbers 12 to 216 in SEQ ID NO: 3, and the other of the primers Corresponding to the second region represented by base numbers 378 to 573 in SEQ ID NO: 1 or 2, or the base regions 374 to 569 in SEQ ID NO: 3, and the first region and the second region The method of claim 5, wherein the sequence between the regions is amplified. 7. The method according to claim 5, wherein the other of the primers corresponds to a region in or near the intervening gene. 8. One of the primers has a base sequence shown in SEQ ID NO: 7, and the other of the primers has a base sequence shown in SEQ ID NO: 8.
The method according to claim 6, which has the nucleotide sequence shown in the following. 9. 18S rDNA and 5.8S rDNA
And the intervening gene (ITS-2) existing between 5.8S rDNA and 28S rDNA (ITS-2), or Ganoderma boninense specific to both genes. A PCR primer consisting of two kinds of oligonucleotides used for identifying or detecting Ganoderma boninense by detecting a sequence, wherein one of the primers has base numbers 12 to 220 in SEQ ID NO: 1 or 2. Or the first region represented by base numbers 12 to 216 in SEQ ID NO: 3, and the other of the primers is represented by base number 3 in SEQ ID NO: 1 or 2.
78-573, or base number 374 in SEQ ID NO: 3.
A primer corresponding to the second region represented by -569, wherein the sequence between the first region and the second region is amplified by PCR using the primer. 10. An 18S rDNA and a 5.8S rDN.
A (ITS-1), an intervening gene (ITS-2) existing between 5.8S rDNA and 28S rDNA, or Ganoderma boninense in both genes. A primer comprising two types of oligonucleotides used for identifying or detecting Ganoderma boninense by detecting a unique sequence, wherein one of the primers has a base number of 12 to 220, base number 12 in SEQ ID NO: 3
To 216, base number 378 to SEQ ID NO: 1 or 2
573 or base numbers 374 to 56 in SEQ ID NO: 3
9. The other of the primers corresponds to the region represented by
A primer corresponding to the intervening gene or a region adjacent thereto.