JP4558371B2 - Primer pair for detecting spinach wilt fungus and molecular identification method using this primer pair - Google Patents

Description

The present invention provides a primer pair for detecting spinach wilt fungus before the occurrence or spread of wilt in spinach cultivation and a molecular identification method using the primer pair for detecting spinach wilt fungus About. Specifically, a primer pair specific to only a specific fungus that causes spinach wilt of Fusarium, and a specific pair of Fusarium that is specific to a specific fungus that causes spinach wilt using this primer pair. The present invention relates to a molecular identification method for spinach wilt fungus, characterized by detecting or quantifying a base sequence.

  Spinach, one of the main crops cultivated in vegetable factories, is often cultivated during the off-season, summer, in order to avoid competition with outdoor products. However, high-temperature and high-humidity breeding is active, and it is easy for diseases caused by fungi that can diffuse in water through conidia (eg Fusarium spp., Psium spp.), Especially due to specific fungus spp. The damage of spinach wilt is serious. When these diseases occur, it is difficult to control while cultivating crops. Therefore, a large loss is required in terms of labor and economy, such as removing spinach during cultivation and total replacement of the culture solution. In order to prevent these diseases, physical sterilizers using ozone and ultraviolet rays used for hydroponics have begun to be sold, but the initial investment cost is very high, so these are introduced and always operated It is not easy. For this reason, it is necessary to constantly monitor the number of pathogenic bacteria in the culture solution so that measures can be taken as appropriate before wilt disease occurs.

  On the other hand, in the method of monitoring the number of bacterial cells, a certain amount of culture solution is sampled and seeded at various dilution concentrations on a medium where only each pathogenic bacteria grows selectively. There is a way to count. However, this method requires a large machine such as a sterilizer or an incubator. It takes 3 to 4 days for isolation of bacteria, and about a week for identification based on morphology and physiology. It is difficult to monitor on a daily basis and in real time. Furthermore, even if a selective medium in which only specific bacteria grow is used, there are more than 100 subspecies of the genus Fusarium that have almost the same morphology and physiology. It is almost impossible to identify. In addition, as an identification method of bacteria, isozyme analysis methods that identify specific strains by extracting specific enzymes from the bacteria and comparing their differences in properties are known, but the time required for identification and the complexity of the operation are known. It doesn't change much, and the separation / identification ability is not so high.

  In recent years, separation and identification have been performed using bacterial genes (DNA). The method using this DNA includes a PCR method in which a specific base sequence is amplified and detected, a DNA extracted from a bacterium is cleaved with a restriction enzyme, and the resulting fragments are electrophoresed. Obtained by performing PCR using the Restriction Fragment Length Polymorphisms (RFLP) method (see Non-Patent Document 1) and unspecified primers, and separating the obtained DNA fragments by electrophoresis in the same manner. For example, there is a RAPD (Random Amplined Polymorphic DNA) method in which discrimination is performed based on differences in patterns. As a method for identifying bacteria by the PCR method, a method using a base sequence of ribosomal DNA is known (see Patent Documents 1 and 2). However, the PCR method presupposes the determination of a specific base sequence that can distinguish the target bacteria from other bacteria, and it is necessary to decode and compare the entire base sequence of rDNA, and the pathogenic type within the same species. This is a problem that the accuracy is inferior to identify.

  In the RFLP method, relatively short DNA such as ribosomal DNA and mitochondrial DNA and polymorphisms associated with specific traits are easily found when the base sequence is known in advance, but large DNA like the genome is analyzed, In the absence of specific primers or probes, it is difficult to detect polymorphisms, and a large amount of highly purified DNA is required for one analysis. In addition, although the RAPD method can easily show polymorphism even with a small amount of DNA, it is not clear whether the obtained polymorphism information is actually related to the target pathogenicity, There are also reproducibility issues in multiple laboratories and numerous tests. In particular, Fusarium spp. Have not yet been found to have a specific nucleotide sequence that can be used for identification at the subspecies level, and spinach wilt fungi have distinct characteristics from other fungi such as specific toxins. This has made it difficult to identify wilt fungi by these methods. For this reason, there is a need for a simple, rapid and highly accurate identification method that enables not only identification of disease-causing bacteria but also monitoring of pathogenic bacteria in culture broth and cultivation soil.

JP-A-10-234381 JP-A-10-234399 Mouyna I. et al., Curr. Genet. 30, p.174-180, 1996

Therefore, the present invention provides a primer pair for detecting only spinach wilt fungus among Fusarium bacteria, and spinach wilt that can identify only spinach wilt fungus among Fusarium fungi in a simple, sensitive and highly accurate manner. It aims at providing the molecular identification method of a fungal fungus.

  As a result of intensive studies to solve the above problems, the present inventors have conducted a comparative analysis of a plurality of Fusarium species including wilt fungi by a RAPD-STS (randomly amplified polymorphic DNA-sequence tagged site) method. Identification and quantification of the target pathogen is achieved with higher sensitivity and accuracy than the PCR method or real-time PCR method using primers designed based on the obtained sequence information after obtaining a nucleotide sequence specific to the wilt fungus. The present invention has been completed.

That is, the present invention
[1] A primer pair for detecting spinach wilt fungus, characterized by comprising a primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 3 ;
[2] Performing PCR or real-time PCR on the test sample subjected to DNA extraction treatment using the primer pair described in [1] above to detect or quantify a base sequence specific to spinach wilt fungus Molecular identification method for spinach wilt fungus
[3] A kit for molecular identification of spinach wilt fungus comprising the primer pair according to [1 ] above;
About.

  The method of the present invention provides accurate information on the presence and the amount of Fusarium oxysporum f.sp. Spinaciae in the culture solution used for hydroponics of spinach, and the method of the present invention. The base sequence to be used is specific for the fungus that causes spinach wilt among many Fusarium spp., And must not cross with non-pathogenic Fusarium spp. Or other fungi. Here, in the molecular identification method, when crossing with non-pathogenic Fusarium genus bacteria and other fungi, it becomes difficult to measure the exact number of wilt disease bacteria in the test sample such as the culture solution. Will give incorrect information in monitoring. Detection of DNA having a specific base sequence can be carried out by a hybridization method, a PCR method using primers designed from sequence information, or a real-time PCR method. The PCR method is preferred. The present invention also includes a method of calculating the number of wilt disease bacteria in a test sample based on the amount of DNA of wilt disease quantified by the above method. The number of bacteria can be easily calculated from the obtained data by preparing a calibration curve between the number of wilt disease bacteria and the amount of DNA to be detected.

ADVANTAGE OF THE INVENTION According to this invention, the primer pair for detection of the spinach wilt fungus of spinach which can identify only the spinach wilt fungus of Fusarium fungi simply, with high sensitivity and high accuracy is provided. This method can easily identify and quantify wilt disease bacteria, and can identify and quantify target bacteria with extremely high sensitivity and high accuracy by combining with PCR or real-time PCR. For this reason, it is very useful because it can be used for routine wilt disease monitoring at a spinach production site and for quick diagnosis / prediction of occurrence at the site.

The meanings of terms used in the present specification are shown below, and the present invention will be described in detail.
In the present specification, unless otherwise specified, the terms `` spinning fungus of spinach '', `` wilt fungus '' and `` Fusarium fungus '' are fungi that cause wilt in spinach among the genus Fusarium (Fusarium oxysporum f.sp. Spinaciae) means fungus.
The method of the present invention detects or quantifies a base sequence specific to spinach dwarf fungus, and is detected or quantified by a hybridization method, a PCR method using primers designed from sequence information, or a real-time PCR method. This specific base sequence is one in which at least 80%, preferably 90% or more, more preferably 95% or more of the base sequences match among wilt fungi. In addition, this specific nucleotide sequence is obtained by culturing a plurality of Fusarium spp., Including wilt fungi, extracting total DNA from these fungi, and subjecting the obtained DNA to the RAPD-STS (randomly amplified polymorphic DNA-sequence). tagged site). In the RAPD-STS method, DNA obtained from a plurality of Fusarium bacteria including wilt fungus is analyzed by RAPD (randomly amplified polymorphic DNA) method to obtain a RAPD marker that can distinguish wilt fungus from other fungi. Based on the base sequence of the RAPD marker, a primer capable of specifically amplifying the RAPD marker is designed.

1. Bacterial culture and DNA extraction Bacterial culture and total DNA extraction can be performed as follows.
The isolated strain is cultured and grown in a medium (for example, potato mode dextrose (PDB) medium) for about 5 to 10 days, and the mycelium of each strain is collected by filtration or the like and freeze-dried. Next, the total DNA is extracted by, for example, CTAB (Cetyl trimethyl ammonium bromide) method. Specifically, a solution containing CTAB is added to the pulverized mycelium and mixed well. After warm bath, add chloroform and mix well and centrifuge at 2500 rpm. After centrifugation, ice-cooled isopropanol is added to the supernatant and kept at −20 ° C. overnight, followed by centrifugation at 2500 rpm. The obtained precipitate is dissolved in a buffer solution, and 2.5 times the amount of ethanol is added, and then centrifuged again at 2500 rpm. The obtained precipitate is dried and then dissolved in a buffer solution or the like. In addition, DNA extraction may be performed using a suitable commercially available kit. Further, the obtained total DNA may be purified before being subjected to the RAPD method.

For example, DNA purification can be performed as follows.
An appropriate amount of cesium chloride is dissolved in the DNA dissolved in the buffer solution, and ethidium bromide is added to perform ultra-far separation at 80000 rpm / 8 hours. Then, DNA bands are collected while visually confirming under ultraviolet irradiation, and an equal amount of isopropanol is added several times to separate and remove ethidium bromide. Salt. Ethanol is added to the obtained solution by 2.5 times volume and centrifuged at 15000 rpm, and the resulting precipitate is dried and then dissolved in a buffer solution to obtain a final DNA solution.

2. RAPD-STS method (1) RAPD marker In PCR reactions to amplify DNA fragments by PCR, the temperature conditions and time of each reaction, the selection and addition amount of primers, etc. are important factors that determine the success or failure of amplification. is there. In particular, the setting of the annealing temperature is particularly important, and the RAPD method is intended to obtain as many DNA fragments as possible by reacting as many unspecified regions and primers as possible. The examination is performed in a temperature range of 30 to 48 ° C., which is 10 ° C. or more lower. Specifically, using the total DNA extracted from each strain as a template, random primers were used for each temperature of denature (94 ° C.), annealing (30-40 ° C.), DNA chain extension (72 ° C.), number of repetitions ( Preliminary PCR is performed 5 to 40 times). The obtained amplification products are subjected to agarose gel electrophoresis and separated, and PCR conditions for obtaining a clear specific amplified fragment are determined. In the present invention, 30 cycles of the PCR reaction are carried out at each temperature and time of denaturer (94 ° C./1 min), annealing (36 ° C./1 min), and DNA strand elongation (72 ° C./1 min). In addition, in order to ensure the deenergization before the start of the reaction cycle, the reaction is performed at 94 ° C./1 min as a preliminary deinert.

  Next, 9 kinds of Fusarium fungi (5 kinds of spinach pathogenic F.o. pv. Spinaciae, 1 kind of non-pathogenic Fusarium F.o. Random primer kit (12mer, A01-) using DNA obtained from F. o. Pv. Batata and two types of tomato pathogenic fusarium F. o. Pv. Licopersici and one type of Pythium ultimum PCR amplification is carried out under the above conditions using H12 primers (96 primers in total, Wako Pure Chemical Industries). Subsequently, the obtained DNA fragments are separated by agarose gel electrophoresis (1% agarose, electrophoresis for 45 minutes at 100 V / constant voltage condition), and an electrophoretic image is taken to detect polymorphism visually. Furthermore, 22 primers out of 96 primers are selected and subjected to PCR amplification, and a band specific to spinach wilt fungus is identified and used as a RAPD marker.

(2) Formation of RAPD-STS The obtained special band (RAPD marker) is cut out from the gel according to a conventional method, then cloned into a plasmid, and the base sequence is determined specifically for spinach wilt fungus. Next, based on the obtained sequence information, a primer for identifying wilt fungus is designed. In addition, the primer design is such that the GC content of the primer, the sense, the value Tm of the antisense primer, the primer does not have a complementary sequence on the 3 ′ end side, the primer does not form a higher order structure, and the total length of the primer. 5 to 50 mer, preferably 15 to 35 mer, a template DNA that lowers the GC content at the 3 ′ end of the primer, avoids a sequence of many identical nucleotides in the primer, and increases the complementarity at the 3 ′ end Note that there are no similar sequences other than the primer region. In general, if the region to be amplified by PCR (template DNA region) is almost the same, even if the primer sequence is shifted by 1 to 10 bases on the 5 ′ side and / or 3 ′ side, the same PCR result (PCR Product) is known to be obtained. Therefore, the primer of the present invention is not limited to a primer having a specific sequence and length, and includes a primer having substantially the same function as the primer. Therefore, in the present invention, for example, in addition to the primer designed as described above, a primer in which one or several bases are deleted, substituted, added and / or inserted, 5 'and / or 3' with respect to the corresponding template DNA. It includes primers that are shifted by 1 or several bases or 10 bases on the side, and primers that match at least 80%, more preferably 90% or more, and even more preferably 95% or more of the original primer.

4). Identification method DNA is extracted from a test sample such as a medium by a conventional method. In this case, what is necessary is just to be able to extract what can be used as a template of PCR amplification, and a commercially available extraction kit etc. may be used. Next, as one embodiment of the present invention, PCR is performed according to a conventional method using the primer of the present invention. At that time, reagents such as a PCR buffer, dNTP, MgCl ₂ may be prepared, or a commercially available PCR kit may be used. PCR conditions include, for example, 40 cycles of 95 ° C./30 seconds + 63 ° C./30 seconds + 72 ° C./30 seconds as one cycle, and finally a termination reaction of 72 ° C. for 7 minutes. The length can be appropriately changed in consideration of the length of DNA to be amplified, the concentration of sample DNA used as a template, and the like. Amplified PCR products can be detected using any method that can identify DNA fragments, specifically agarose gel electrophoresis, acrylamide gel electrophoresis, capillary electrophoresis, hybridization, immunological methods, etc. it can. In general, the PCR product is subjected to electrophoresis on an agarose gel to detect a band.

  In one embodiment of the present invention, the wilt fungus may be identified by a real-time PCR method. The real-time PCR method is a method for monitoring and analyzing the amount of PCR amplification in real time, and does not require electrophoresis and is excellent in rapidity and quantitativeness. In this method, a standard curve is prepared by performing PCR using a known amount of DNA that has been serially diluted in advance as a standard. PCR is also performed on the test sample under the same conditions, and the detection and concentration of DNA to be detected in the sample are measured. In the present invention, the real-time PCR method, for example, is carried out 40 cycles of 95 ° C./10 seconds + 53 ° C./15 seconds + 68 ° C./30 seconds after the following conditions: 95 ° C./600 seconds hold. The length can be appropriately changed in consideration of the length of DNA to be amplified. The present invention can also easily determine the bacterial count of the wilt fungus by correlating the calibration curve with the bacterial count of the wilt fungus.

The present invention includes a kit containing a primer used in the above-described detection or quantification method. In addition to primers, the kit includes other reagents such as dNTP, MgCl ₂ , Taq DNA polymerase and other polymerases, fluorescent reagents, buffers, glycerol, DMSO, positive control DNA, negative control DNA, distilled water, and the like. May be. These reagents may be individually packed in the kit, or may be a mixture of two or more reagents. In addition, information such as suitable PCR conditions may be attached to the kit, or only the primer reagent may be included.

[Example]
An embodiment of the present invention will be described below with reference to FIG.
1. Strains and Cultures In this example, nine kinds of Fusarium spp. Shown in Table 1 (5 spinach wilt fungi with different origins, 1 non-pathogenic fusarium, 1 kind of sweet potato pathogenic fusarium, and Two types of tomato pathogenic fusarium) and one type of Pythium ultimum were used. These strains were cultured in 250 ml of potato dextrose (PDB) medium at 28 ° C. at 125 rpm in the dark. The mycelium of each strain was concentrated with Toyo Denshi No5B.

2. RAPD-STS
Above 1. The cells collected in (1) were pre-frozen at −80 ° C. and then freeze-dried. This was ground into a powder using a mortar and pestle. Next, DNA was extracted using a commercially available kit (ISOPLANT kit, NIPPON GENE, Toyama, Japan). For PCR, the total reaction volume was 50 μl, 100 ng of extracted genomic DNA and 20 ng of primer were added, and a commercially available PCR reagent (Super Taq Premix Kit, Sawady Tec. Inc.) was used. PCR was performed using 96 types of primers (12 mer, random primer, Wako Pure Chemical Industries) as the primers, and 22 types of primers shown in Table 2 were selected. The PCR conditions were 94 ° C./1 min initial deigner, 94 ° C./1 min + 36 ° C./1 min + 72 ° C./1 min, 30 cycles, and final extension reaction 72 ° C./7 min.

  The DNA fragments obtained by the above PCR amplification were separated by agarose gel electrophoresis using a mini-submarine type electrophoresis tank (mupid, Cosmo Oil Co., Ltd.) (1% agarose, 100V / constant voltage condition for 45 minutes) ), A polymorphism was detected visually by photographing an electrophoretic image. Among the detected bands, 66 polymorphic fragments serving as RAPD markers were analyzed in a dendritic manner for each bacterial species using software (UPGMA analysis, Genetyx-Win ver.5). However, it was classified into five clusters as shown in FIG. Cluster 1 is Psium, Cluster 2 is non-pathogenic among F.o. pv. Spinaciae, Cluster 3 is Fusarium that is pathogenic to tomatoes, Cluster 4 is Fusarium that is pathogenic to sweet potato, Cluster 5 was a Fusarium genus having pathogenicity to spinach. In addition, although these clusters are clearly classified into five, it was shown that each genetic closeness is very close.

In the PCR amplification, primers C05 (SEQ ID NO: 9), C09 (SEQ ID NO: 10), C11 (SEQ ID NO: 11), D03 (SEQ ID NO: 12), F10 (SEQ ID NO: 22) and G11 (SEQ ID NO: 24) were used. When used, a clear band (RAPD marker) specific for spinach wilt fungus was obtained. Thirty This resulting specific RAPD marker, was cut out from the gel according to a conventional method, was cloned into plasmid ^{pCR (TOPO (R) vector,} TOPO AT Cloning (R) kit) to determine the nucleotide sequence (ABI PRIMS ^TM 3100 Genetic Analyzer, Perkin-Elmer). Here, based on the base sequence of the RAPD marker (SEQ ID NO: 1) obtained from the C05 primer, a primer for identifying spinach wilt fungus was designed. For the designed primers, the reproducibility of specific fragments was confirmed by PCR. As a result, PCR was performed using a set of primers C05-F (SEQ ID NO: 2) and C05-R (SEQ ID NO: 3), and the reproducibility of specific PCR amplification for identifying spinach wilt fungi Confirmed (see FIG. 2).

3. Real-time PCR
Real-time PCR was performed as follows using the C05-F and C05-R primer sets.
All reactions a total reaction volume ^{25μl (12.5μl SYBR (R) Green} , 2 × PCR Master Mix (Applied Biosystems), genomic DNA 2 ng, 0.3 [mu] M primers, sterile distilled water) was. For real-time PCR, Takara Bio's Smart Cycler ^(R) was used, and after 95 ° C./600 seconds hold, the reaction was carried out under conditions of 40 cycles of 95 ° C./10 seconds + 53 ° C./15 seconds + 68 ° C./30 seconds. As a result, as confirmed by normal PCR, only the spinach pathogenic Fusarium 5 strains yielded positive amplification products, but other strains were not amplified (see FIG. 3).

4). Sensitivity and Quantitative Analysis The sensitivity of spinach wilt fungus identification was verified by normal PCR and real-time PCR for the C05-F and C05-R primer sets of the present invention. As a result, 10 to 100 pg of DNA was required for normal PCR (FIG. 4), but the real-time PCR method could be identified if there was only 1 pg of wilt of the wilt disease (FIG. 5). Furthermore, when the quantitative property of real-time PCR was examined, a strong positive correlation was observed between the number of cycles and the DNA (logarithmic value) used as a template (R ² >0.99; FIG. 6). It was confirmed that by combining the primer of the present invention and the real-time PCR method, an excellent discrimination result can be obtained in terms of both quantitativeness and sensitivity.

It is a figure which shows the result of carrying out the dendritic analysis of the genetic relatedness of each microbial species. It is a figure which shows the result of having detected the wilt fungus of spinach specifically by PCR. It is a figure which shows the result of having specifically detected spinach wilt fungus by real-time PCR. It is an electrophoresis image which shows the result of verification of the detection sensitivity in PCR. It is a graph which shows the result of verification of detection sensitivity in real-time PCR. It is a graph which shows the correlation of the cycle number in real time PCR, and template DNA.

Claims (3)

A primer pair for detecting spinach wilt fungus, characterized by comprising a primer consisting of the base sequence shown in SEQ ID NO: 2 and a primer consisting of the base sequence shown in SEQ ID NO: 3 . A spinach characterized by detecting or quantifying a base sequence specific to spinach wilt fungus by performing PCR or real-time PCR on a test sample subjected to DNA extraction treatment using the primer pair according to claim 1 Molecular identification method for wilt fungus . A kit for molecular identification of spinach wilt fungi comprising the primer pair according to claim 1 .