WO2022102726A1 - Procédé de diagnostic du sol fusarium oxysporum par génomique du pathogène - Google Patents

Procédé de diagnostic du sol fusarium oxysporum par génomique du pathogène Download PDF

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WO2022102726A1
WO2022102726A1 PCT/JP2021/041612 JP2021041612W WO2022102726A1 WO 2022102726 A1 WO2022102726 A1 WO 2022102726A1 JP 2021041612 W JP2021041612 W JP 2021041612W WO 2022102726 A1 WO2022102726 A1 WO 2022102726A1
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specific
host
nucleotide sequence
fusarium
differentiated
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Japanese (ja)
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秀太 浅井
パメラ ガン
幸子 増田
侑 鮎川
賢 白須
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国立研究開発法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to a method, a kit, etc. for detecting Fusarium bacterium, and specifically, to a method, a kit, etc. for detecting a host-specific differentiated Fusarium bacterium.
  • Fusarium oxysporum which causes serious soil diseases all over the world, has more than 120 types of host-specific differentiated types (hereinafter referred to as “Fusarium oxysporum”) that cause soil diseases using different plant species as specific hosts. , Also referred to as “forma specialis”) is known to exist. Fusarium bacterium carries a gene involved in pathogenicity on a small chromosome, and it is considered that host specificity is determined by this (Non-Patent Document 1). However, since little is known about the structure and function of small chromosomes, it has not been possible to evaluate the differentiated type of Fusarium fungus based on DNA analysis.
  • the subject of the present invention is a differentiated type fusalium fungus that cannot be evaluated by conventional DNA analysis techniques, particularly a differentiated type that causes soil diseases using high commercial value plants such as Abrana family plants, lettuce, spinach and banana as specific hosts. It is to develop a technology that enables trace detection of Fuzarium fungi.
  • a further object of the present invention is to detect trace amounts of at least two types of differentiated Fusarium bacteria having different host specificities among the differentiated Fusarium bacteria that cause soil diseases using the plant having high commercial value as a specific host. It is to develop a technology that enables a mixed solution.
  • the present inventors have pursued diligent research focusing on differentiated Fusarium fungi that cause soil diseases using Abranaceae plants, lettuce, spinach or banana as a specific host, and determined the entire genome sequence of a novel Fusarium strain. As a result of comparative analysis, the present invention was completed by finding a unique genomic sequence that is commonly present in differentiated Fusarium fungi having the same host specificity.
  • the present invention provides a method for detecting a host-specific differentiated Fusarium bacterium.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention is a Fusarium bacterium that causes soil disease using an Abrana family plant as a specific host, a Fusarium bacterium that causes soil disease using lettuce as a specific host, and a spinach.
  • the DNA having the nucleotide sequence contained in Fusarium fungus is contained in Fusarium fungus using a set of primers having a part of the nucleotide sequence and a part of the nucleotide sequence complementary to the nucleotide sequence, a probe or a single-stranded circular template. Includes the step of detecting from the sample.
  • the differentiated-type specific genomic region of Fuzarium bacterium that causes soil disease using the Abranaceae plant as a specific host contains the nucleotide sequence of SEQ ID NO: 1 and is lettuce.
  • the differentiation-specific genomic region of Fuzarium bacterium that causes soil disease using the specific host contains the nucleotide sequence of SEQ ID NO: 2, and the differentiation-specific genome of Fuzarium bacterium that causes soil disease using spinach as a specific host.
  • the region comprises the nucleotide sequence of SEQ ID NO: 3, and the differentiation-specific genomic region of Fusalium bacterium that causes soil diseases using banana as a specific host can contain the nucleotide sequence of SEQ ID NO: 4.
  • the set of primers contains an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 6, or the probe or single strand.
  • Circular templates can include the nucleotide sequence of SEQ ID NO: 5 or 6 and When the specific host is lettuce, the set of primers comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 8, or the probe or the single-stranded ring.
  • the template can contain the nucleotide sequence of SEQ ID NO: 7 or 8.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 10, or the probe or single-stranded circular template.
  • the set of primers comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 11 and a nucleotide of SEQ ID NO: 12.
  • the probe or single-stranded circular template may include the nucleotide sequence of SEQ ID NO: 11 or 12 or include an oligonucleotide containing the sequence.
  • the primer set is a pair of primers consisting of two oligonucleotides, each consisting of a sequence of 15 to 25 nucleotides in length.
  • One primer of the pair of primers contains a part of the nucleotide sequence of the differentiation-specific genomic region, and the other primer of the pair of primers contains a nucleotide sequence complementary to the nucleotide sequence of the differentiation-specific genomic region.
  • the nucleotide sequence complementary to the nucleotide sequence of the other primer is from the position of the nucleotide corresponding to the nucleotide at the 5'end of the nucleotide sequence of the other primer.
  • the length to the position of the nucleotide corresponding to the nucleotide at the 3'end of the nucleotide can be 100 to 500 nucleotides.
  • the set of primers can be a pair of two primers.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10 and / or.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12.
  • At least one of the primer set and the probe can be labeled with a detectable atomic group.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention is a Fusarium bacterium that causes soil disease using an Abrana family plant as a specific host, a Fusarium bacterium that causes soil disease using lettuce as a specific host, and a spinach. At least two types of Fusarium fungi with different host specificity selected from the group consisting of Fusarium fungi that cause soil diseases with banana as a specific host and Fusarium fungi that cause soil diseases with banana as a specific host. A step of simultaneously detecting differentiated Fusarium fungi in the same reaction mixture can be included.
  • the Brassicaceae plant can be selected from cabbage, radish, komatsuna and stock.
  • the sample can be a soil sample.
  • the present invention provides a soil diagnostic method for host-specific differentiated Fusarium fungi.
  • the method for soil diagnosis of the host-specific differentiated Fusarium bacterium of the present invention uses the method for detecting the host-specific differentiated Fusarium bacterium of the present invention using the sample as a soil sample, and the host-specific differentiated Fusarium bacterium in the soil sample. And when the differentiated Fusarium bacterium is detected in the soil sample by the step, it is diagnosed that the soil sample contains the differentiated Fusarium bacterium, or in the soil sample by the step. When the differentiated Fusarium bacterium is not detected, the soil sample comprises a step of diagnosing that the differentiated Fusarium bacterium is not contained.
  • the present invention provides a kit for carrying out the method for detecting the host-specific differentiated Fusarium fungus of the present invention, or a kit for carrying out the soil diagnosis method for the host-specific differentiated Fusarium fungus of the present invention.
  • the kit of the present invention comprises a set of primers, a probe or a single-stranded circular template used in the method for detecting differentiated Fusarium fungi of the present invention and / or the method for soil diagnosis of differentiated Fusarium fungi of the present invention.
  • Fusarium fungus that causes soil disease using the Abrana family plant as a specific host Fusarium fungus that causes soil disease using lettuce as a specific host, and Fusarium fungus that causes soil disease using spinach as a specific host.
  • the differentiation-specific genomic region of Fusarium fungus with banana as a specific host contains the DNA of the nucleotide sequences of SEQ ID NOs: 1, 2, 3 and 4, respectively.
  • the primer set is a pair of primers consisting of two oligonucleotides, each consisting of a sequence of 15 to 25 lengths.
  • One primer of the pair of primers contains a part of the nucleotide sequence of the differentiation type-specific genomic region of any one of SEQ ID NOs: 1, 2, 3 or 4, and the other primer of the pair of the primers contains the differentiation-specific specificity. Containing a portion of a nucleotide sequence complementary to the nucleotide sequence of a genomic region, and Of the double-stranded DNA of the differentiation-specific genomic region, the nucleotide sequence complementary to the nucleotide sequence of the other primer is from the position of the nucleotide corresponding to the nucleotide at the 5'end of the nucleotide sequence of the other primer.
  • the length to the position of the nucleotide corresponding to the nucleotide at the 3'end of the nucleotide can be 100 to 500 nucleotides.
  • the set of primers contains an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 6, or the probe or single strand.
  • Circular templates can include the nucleotide sequence of SEQ ID NO: 5 or 6 and
  • the set of primers comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 8, or the probe or single-stranded circular template.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 10, or the probe or single-stranded circular template.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 12, or the probe or single-stranded circular template.
  • At least one of the primer set and the probe can be labeled with a detectable atomic group.
  • the Brassicaceae plant can be selected from cabbage, radish, Japanese mustard spinach, stock and other Brassicaceae plants.
  • the bacteria whose genome has been newly decoded are described in the remarks column.
  • the assembly accession number of NCBI GenBank is described in the origin column.
  • Forward primer (SEQ ID NO: 5) common to bacteria hosting any of Brassicaceae cabbage, radish or komatsuna, and reverse primer common to bacteria hosting brassicaceae cabbage, radish or komatsuna (SEQ ID NO: 6) ) Is underlined.
  • Part of the nucleotide sequence of the genomic region specific to lettuce (SEQ ID NO: 2).
  • the positions of the complementary sequences of the lettuce forward primer (SEQ ID NO: 7) and the lettuce reverse primer (SEQ ID NO: 8) are underlined.
  • Part of the nucleotide sequence of the genomic region specific to spinach (SEQ ID NO: 4).
  • the positions of the complementary sequences of the spinach forward primer (SEQ ID NO: 9) and the spinach reverse primer (SEQ ID NO: 10) are underlined.
  • the positions of the complementary sequences of the banana fungus forward primer (SEQ ID NO: 11) and the banana fungus reverse primer (SEQ ID NO: 12) are underlined.
  • a panel of electrophoretograms showing the detection of Fusarium fungi by the PCR method using the common differentiation-specific primer pair of Brassicaceae and the common primer pair of Fusarium fungi of the present invention.
  • the electrophoretogram on the panel shows differentiated Fuzarium fungi hosted by Brassicaceae cabbage, daikon and Komatsuna, and differentiated Fuzarium fungi hosted by other Brassicaceae plants (Stock fungus (1) and (2)), Agarose gel electricity of the reaction product obtained as a result of PCR reaction using a sample of banana fungus, cucumber fungus and non-pathogenic Fuzarium fungus and a pair of differentiation type-specific primers common to Brassicaceae fungi. The migration band is shown.
  • the electrophoretogram below the panel shows the Fuzarium fungus extracellular matrix protein 1 (FoFEM1) constitutively expressed in all Fuzarium fungi reported by Michiels et al.
  • FoFEM1 extracellular matrix protein 1
  • the table which shows the result of having performed the detection test of the Fusarium strain by the PCR method using the differentiation type specific primer pair of this invention.
  • Each column of the table in FIG. 8 shows a pair of primers (differentiation-specific primers for spinach, lettuce, banana and Brassicaceae, and common primers for Fusarium) used in the PCR detection test, and each primer.
  • the specific detection accuracy of the detection test using pairs is shown. The specific detection accuracy is expressed as a percentage of the number of strains obtained by subtracting the number of false-negative and false-positive differentiated strains from the total number of differentiated strains tested (146 strains).
  • Each row in the table shows the sample Fusarium strains (lettuce, onion, spinach, pea, other legumes, bananas, tomatoes, cucumbers, melons, other cucurbitaceae, cabbage, etc. Shows cucurbita, lettuce, stock and non-pathogenic bacteria).
  • the denominator of the fractional notation in the table of FIG. 8 indicates the number of differentiated Fusarium strains of the sample in each row, and the numerator indicates the number of positive differentiated Fusarium strains.
  • positive means that the band of DNA amplified by the PCR reaction could be detected by agarose gel electrophoresis.
  • Electrophoresis showing the results of multiplex PCR reaction on a mixed sample of cabbage, spinach, lettuce and banana using a primer pair specific for cabbage, spinach, lettuce or banana of the present invention.
  • FIG. 9 represent lanes of PCR reaction products using primer pairs and cell samples specific to each, and “Mixture (4). Strain) ”represents a lane of PCR reaction products using four differentiated specific primer pairs and cell samples.
  • Electrophoretic diagram panel The electrophoretogram on the panel of FIG. 10 was obtained as a result of performing a PCR reaction using a soil sample of artificially contaminated soil supplemented with onion, cabbage or spinach and a pair of common primers for Brassicaceae. The agarose gel electrophoresis band of the reaction product is shown.
  • FIG. 10 shows the results of PCR reaction using a soil sample of artificially contaminated soil supplemented with onion, cabbage or spinach and a pair of differentiation-specific primers of spinach.
  • the agarose gel electrophoresis band of the obtained reaction product is shown.
  • the electrophoretic diagram at the bottom of the panel of FIG. 10 shows the reaction obtained as a result of performing a PCR reaction using a soil sample of artificially contaminated soil supplemented with onion fungus, cabbage fungus or spinach fungus and a pair of Fuzarium fungus common primers.
  • the agarose gel electrophoresis band of the product is shown.
  • An electrophoretogram showing the detection sensitivity of spinach in soil using the spinach primer pair of the present invention.
  • Agarose gel electrophoresis was used to detect the reaction product obtained by performing a PCR reaction using a part of the soil inoculated with 10 to 106 differentiated Fusarium spores using spinach as a specific host per 1 g of soil as a sample.
  • “Mock” represents the lane of the PCR reaction product of the soil sample to which the spore was not added
  • " 101 " to " 106 " represent the lane of the PCR reaction product of the soil sample having the spore concentration.
  • M represents a lane of standard polynucleotide markers comprising 100 bp, 200 bp and 300 bp.
  • One embodiment of the present invention is a method for detecting a host-specific differentiated Fusarium bacterium.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention is a Fusarium bacterium that causes soil disease using an Abrana family plant as a specific host, a Fusarium bacterium that causes soil disease using lettuce as a specific host, and a spinach.
  • the DNA having the nucleotide sequence contained in Fusarium fungus is contained in Fusarium fungus using a set of primers having a part of the nucleotide sequence and a part of the nucleotide sequence complementary to the nucleotide sequence, a probe or a single-stranded circular template. Includes the step of detecting from the sample.
  • the method for detecting host-specific differentiated Fusarium fungi of the present invention causes soil diseases using Fusarium fungi as a specific host and lettuce as a specific host.
  • DNA having a nucleotide sequence contained in a type-specific genomic region is subjected to a set of primers having a part of the nucleotide sequence and a part of the nucleotide sequence complementary to the nucleotide sequence, a probe or a single-stranded circular template.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention includes a Fusarium bacterium that causes soil disease using an Abrana family plant as a specific host and a Fusarium bacterium that causes soil disease using lettuce as a specific host.
  • At least three types of Fusarium bacterium selected from the group consisting of Fusarium bacterium that causes soil disease using spinach as a specific host and Fusarium bacterium that causes soil disease using banana as a specific host.
  • Fusarium fungi using DNA having a nucleotide sequence contained in a genomic region, using a set of primers having a part of the nucleotide sequence and a part of a nucleotide sequence complementary to the nucleotide sequence, a probe or a single-stranded circular template. May include a step of detecting from a sample containing.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention includes a Fusarium bacterium that causes soil disease using an Abrana family plant as a specific host and a Fusarium bacterium that causes soil disease using lettuce as a specific host. , Fusarium fungi that cause soil diseases with spinach as a specific host and Fusarium fungi that cause soil diseases with banana as a specific host.
  • a step of detecting the DNA contained in a sample containing Fusarium fungus using a set of primers having a part of the nucleotide sequence and a part of the nucleotide sequence complementary to the nucleotide sequence, a probe or a single-stranded circular template. May include.
  • the soil disease is a disease caused by the invasion and proliferation of the pathogen Fuzarium fungus that survives in the soil from the roots, underground stems, stems and other parts of the plant in contact with the soil, and is caused by wilting, withering and root rot. And other illnesses.
  • Fusarium bacterium refers to the Fusarium oxysporum species in the genus Fusarium, which is an ascomycete fungus. Fusarium fungi are distributed in soils and plants all over the world, and cause wilting, withering, root rot, etc. in many host crops. Fuzarium is a root-based living bacterium that invades the host when it wakes up from dormancy in the root sphere of the host plant, but in many cases the saprophytic life after the host's death is limited to the residue of its own host. The host specificity of the infected plant is determined for each strain.
  • the "host-specific differentiated type or differentiated type (forma specialis)" of Fusarium fungus refers to a classification category lower than the species of Fusarium fungus. It is known that the range of plant species to be infected, that is, the host specificity of Fusarium fungi is determined for each differentiated type, and that there are more than 120 types of Fusarium fungi. In the present specification, the distinction by the host plant specific to each differentiated type is expressed by abbreviating the differentiated type of Fusarium fungus having cabbage as a specific host, for example, as "cabbage fungus".
  • the host is based on the sequence data of the differentiated fusalium strain whose whole genome sequence has been decoded. Attempts have been made to discover unique nucleotide sequences that are common to all differentiated fusalium strains of the same specificity but do not exist in other differentiated fusalium strains.
  • FIG. 1 is a table showing the scientific name, strain name, origin, etc. of the Fusarium bacterium of the present invention.
  • the bacteria whose genome has been newly decoded are described in the remarks column of FIG.
  • the assembly accession number of NCBI GenBank is described in the origin column.
  • the differentiated Fusarium strains in which the entire genome sequence has been newly decoded in the present specification are 10 types of strains specified as "new genome decoding strains" in the remarks column.
  • the differentiated Fusarium strains used for analysis in the present specification whose entire genome sequence has already been decoded by a third party, are four types of strains in which a code number starting with "GCA" is specified in the origin column of FIG. Is.
  • the table in FIG. 2 shows the estimated genomic size of these strains, the size of the differentiation-specific genomic region candidate sequence, and the genomic region common to cabbage, radish, and komatsuna (hereinafter, "common to Brassicaceae”).
  • the size of the candidate sequence and the candidate sequence of the genomic region common to cabbage and melon (hereinafter referred to as “common to Brassicaceae) is shown.
  • FIG. 2 is a table showing 10 types of strains in which the entire genome sequence was newly decoded in the present invention and 4 types of strains in which the entire genome sequence was already decoded used in the comparative analysis in the present invention.
  • the table of FIG. 2 shows the estimated genome size of these strains, the size of the differentiation-specific genomic region candidate sequence, and the size of the Brassicaceae common candidate sequence and the Cucurbitaceae common candidate sequence.
  • genomic DNA refers to genetic information on all nucleic acids possessed by a certain Fusarium bacterium. Van Dam, P.M. According to (Scientific Rep. 7: 9042 (2017)), Fusalium bacterium contains about 11 core chromosomes and one or more small chromosomes (accessory chromosomes (s)), and exhibits host specificity. The defining gene is located on a small chromosome. Therefore, the entire genome sequence of Fusarium bacterium refers to the nucleotide sequence of all nucleic acids of the core chromosome and the small chromosome.
  • the differentiation-specific genomic region is commonly present in a unique nucleotide sequence for each of the 14 types of whole genome sequences, that is, in strains of differentiated Fusalium bacteria having the same host specificity. However, it refers to a polynucleotide consisting of a nucleotide sequence that does not exist in the strains of differentiated Fuzarium bacteria having different host specificities.
  • the sample containing Fusarium fungus refers to a sample containing Fusarium fungus isolated and cultured or Fusarium fungus derived from nature.
  • the sample containing Fusarium bacterium is any liquid medium that can be used for culturing Fusarium bacterium, a buffer capable of stably holding the DNA of Fusarium bacterium, for example, TE buffer (10 mM Tris-HCl (pH: 7.4 to 8.). It may be suspended in an aqueous solution containing, but not limited to, 0) and 1 mM EDTA).
  • PDA potato dextrose agar
  • potato dextrose liquid medium obtained by removing agar from PDA medium Komada medium and other media
  • the PDA medium is a medium in which 4.0 g of potato extract, 20.0 g of glucose and 15.0 g of agar are dissolved in 1 L of pure water, sterilized by high-pressure steam in an autoclave, and then dispensed into a petri dish and coagulated. If necessary, add 0.1 g / 1 L of chloramphenicol.
  • Fusarium bacteria can be collected by statically culturing them in a PDA medium at 28 ° C. to isolate colonies, and culturing them in a potato dextrose liquid medium at 28 ° C. for 5 days with shaking. The recovered cells were frozen in liquid nitrogen and then crushed in a mortar until they became powdery.
  • G. And Thomasson, W.M. F. Genomic DNA can be prepared according to (Nucleic Acid Research, 8: 4321 (1980)).
  • the step of detecting a DNA having a nucleotide sequence contained in the differentiation-specific genomic region from a sample containing Fusalium bacterium is specificizing a nucleic acid encoding a part of the unique nucleotide sequence from the sample. Amplification is performed to determine the presence or absence of the nucleic acid.
  • Specific amplification of a nucleic acid encoding a portion of the unique nucleotide sequence from the sample refers to an oligonucleotide comprising a nucleotide sequence containing the 5'end of a portion of the unique nucleotide sequence and the unique nucleotide sequence.
  • an oligonucleotide containing a nucleotide sequence containing the 5'end of a part of the complementary sequence as a primer an amplification reaction with a temperature cycle and / or an isothermal amplification reaction without a temperature cycle can be performed, and the unique nucleotide can be used.
  • RCA rolling circle amplification
  • Amplification reactions involving the temperature cycle include polymerase chain reaction (PCR), TaqMan® assay, polymerase-mediated amplification reaction (helicase-dependent amplification (HDA), ligase-polymerase amplification (RPA), and rolling circle amplification. (RCA) etc.), both ligase-mediated amplification reactions (such as ligase detection reaction (LDR), ligase chain reaction (LCR), and their respective gap types), and combinations of nucleic acid amplification reactions such as LDR and PCR. , Not limited to these.
  • the temperature cycle of the amplification reaction with a temperature cycle is designed to allow the first denaturation step at high temperature, followed by the template denaturation step, the primer annealing step, and the polymerase extension of the annealed primer. Includes repeated temperature cycles.
  • the sample is first heated at a temperature of about 95 ° C. for about 2-10 minutes to denature the double-stranded DNA sample.
  • the sample is then denatured for about 10-60 seconds at the beginning of each cycle, depending on the type of sample and equipment used.
  • the primer is annealed to the target DNA at a lower temperature of about 40 ° C to about 60 ° C for about 20 to 60 seconds.
  • Primer extension by polymerases is often carried out at temperatures ranging from about 60 ° C to about 72 ° C.
  • the amount of time used for elongation depends on the size of the amplicon and the type of enzyme used for amplification and is readily determined by routine experimentation.
  • the annealing process can be combined with the extension process, resulting in a two-step cycle.
  • the temperature cycle may include further temperature shifts.
  • the number of cycles used in the amplification reaction depends on many factors, including the primers used, the amount of sample DNA present, and the temperature cycle conditions. The number of cycles to be used for any amplification reaction can be readily determined by one of ordinary skill in the art using routine experiments.
  • a final extension step may be added after the temperature cycle is completed to ensure the synthesis of all amplification products. Further, for storage of the reaction product, the reaction product can be stored at 4 ° C. after all the reaction steps are completed.
  • Amplification reactions involving temperature cycles can be performed using any device known to those of skill in the art. These devices include thermal cyclers that can set the temperature, reaction time and number of cycle cycles for temperature cycle iterations, as well as devices that include thermal cyclers, ray emitters, and fluorescent signal detectors. It is commercially available.
  • HDA helicase-dependent amplification
  • RPA recombinase polymerase amplification
  • SDA chain substitution
  • LAMP loop-mediated isothermal amplification
  • RCA rolling circle amplification
  • the HDA method and the RPA method include a forward primer containing the 5'-terminal nucleotide sequence of the nucleotide sequence to be amplified (hereinafter referred to as "target sequence").
  • target sequence Two primers with a reverse primer containing the complementary sequence of the nucleotide sequence at the 3'end of the target sequence are required.
  • the SDA method requires four primers.
  • the LAMP method requires 4 to 6 primers containing the nucleotide sequence inside the target sequence.
  • the RCA method requires a single-stranded circular template containing one nucleotide sequence in the target sequence or a paddock probe which is a single-stranded linear DNA of the precursor thereof.
  • Performing a rolling circle amplification (RCA) reaction using a single-stranded circular template containing a portion of the unique nucleotide sequence is a single-stranded circular containing a complementary sequence of a portion of the nucleotide sequence of the genomic sequence of the Fusalium strain.
  • a step of forming a complex in which the single-stranded DNA complementary to the single-stranded circular template of the genomic DNA of the Fusalium strain forms a pair with the single-stranded circular template, and a DNA polymerase (for example, for example) in the complex. Includes a step of reacting a ⁇ 29 DNA polymerase) with a deoxyribonucleoside substrate to synthesize a single-stranded linear DNA in which the complementary sequence of the single-stranded circular template is repeatedly continuous (Nisson, M. et al., Science, 265: 2085 (Nilsson, M. et al., Science, 265: 2085). 1994)).
  • An alternative procedure for the RCA reaction is to cleave part of the unique nucleotide sequence in two, including the 5'side nucleotide sequence on the 3'side and the 3'side nucleotide sequence on the 5'side.
  • the paddock probe is combined with the fuzarium bacterium genomic DNA containing a part of the unique nucleotide sequence.
  • a step of forming a hybrid a step of adding T4DNA ligase to connect the 3'end and the 5'end of the paddock probe with a phosphodiester bond to form a single-stranded cyclic template, and a DNA polymerase to the complex.
  • T4DNA ligase to connect the 3'end and the 5'end of the paddock probe with a phosphodiester bond to form a single-stranded cyclic template
  • a DNA polymerase for example, ⁇ 29 DNA polymerase
  • a deoxyribonucleoside substrate are reacted with each other to synthesize a single-stranded linear DNA in which the complementary sequence of the single-stranded circular template is repeatedly continuous (Nilsson, M. et al., Science, 265). : 2085 (1994)).
  • Improvements to the RCA method include, for example, the circle-to-circle (C2CA) method (Dahl, F.
  • a single-stranded linear DNA synthesized using the single-stranded circular template as a template is paired with an oligonucleotide containing a specific restriction enzyme recognition sequence, and the restriction is performed. It comprises the step of cutting the single-stranded linear DNA into fragments of the size of the single-stranded circular template by cleaving with an enzyme.
  • the C2CA method further comprises a step of inactivating the restriction enzyme at a high temperature and then lowering the temperature to form a hybrid with the newly generated single-stranded linear / BR> CNA fragment in which the rest of the oligonucleotide is generated, and T4DNA. It comprises the step of adding a ligase to connect the 3'end and the 5'end of the paddock probe with a phosphodiester bond to form a single chain cyclic template.
  • the C2CA method is more exponential than simple rolling circle DNA synthesis by repeating rolling circle DNA synthesis from the single-stranded circular template and single-stranded circular template formation from the obtained single-stranded linear DNA.
  • the unique nucleotide sequence of the target can be amplified.
  • each primer should be 15 to 25 nucleotides.
  • the number of bases in the sequence amplified by the primer is 100 to 500.
  • the GC content of each primer is 40 to 60% or 45 to 55%.
  • the Tm value of each primer should not differ significantly. Alternatively, the difference between the Tm values of the forward primer and the reverse primer is within about 10%, within about 9%, within about 8%, within about 7%, within about 6%, and about 5% of the Tm value of either primer. Within, within about 4%, within about 3%, within about 2%, or within about 1%. -There is no base bias in the sequence of each primer.
  • -It is better to avoid whether the 3'end sequence of the primer is a sequence rich in guanine and cytosine or a sequence rich in adenine and thymine, and avoid the 3'end base from thymine.
  • BLAST, FASTA, etc. homology search software
  • Primers for the PCR method are known to those skilled in the art as paid or free primer design software (eg, OLIGO® Primer Analysis Software (Molecular Biology Industries, Inc. (Colorado Spring, USA)). , Primer, A. et al.).
  • the primers can be designed to meet the requirements similar to the requirements for designing the primers of the PCR method.
  • the presence or absence of is detected by the step of intercalating a fluorescent dye with amplified nucleic acid, particularly double-stranded DNA, and the presence or absence of a band of amplified nucleic acid separated by electrophoresis or the fluorescence intensity of the band.
  • an arm consisting of a nucleotide sequence having complementation is arranged on the 5'side and the 3'side of the complementary sequence of the amplified nucleic acid, and two arms sandwiching the complementary sequence of the amplified nucleic acid are paired with each other.
  • a step of preparing a probe in which a fluorescent reporter molecule and an extinguishing molecule capable of extinguishing the fluorescence of the reporter molecule are bound to each of the 5'side end and the 3'end to form a hairpin loop structure, and a step of preparing a target nucleic acid.
  • the hairpin loop structure is broken and the two arms are separated, and the extinct molecule is separated from the fluorescent reporter molecule according to the amplification amount of the target nucleic acid. Includes a step of detecting fluorescence.
  • improved PCR methods such as the quantitative real-time PCR method, in which the amplified target nucleic acid is quantified at the same time as the amplification, are well known to those skilled in the art.
  • the fluorescent dye used in the step of intercalating the fluorescent dye with the amplified nucleic acid, particularly double-stranded DNA includes, but is not limited to, ethidium bromide, SYBR Green and TB Green.
  • the fluorescent reporter molecule and the corresponding quenching molecule have, for example, the following combinations (1) to (3).
  • 6-carboxyfluorescein (6-FAM TM), 6-carboxy-4', 5'-dichloro-2', 7'-dimethoxyfluorescein (JOE TM), 6-carboxy-1,4 -From dichloro-2', 7'-dichlorofluorescein (TET TM) and 6-carboxy-1,4-dichloro-2', 4', 5', 7'-tetrachlorofluorescein (HEX TM)
  • Quenching molecules for fluorescent reporter molecules selected from the group are BHQ-1 and / or DABCYL.
  • Quenching molecules for fluorescent reporter molecules selected from Cyanine 3, 6-carboxy-X-Rhodamine (ROX TM) and TxRd (Sulfurhodamine 101-X) are BHQ-2, DABCYL.
  • the quenching molecule for the fluorescent reporter molecule selected from the group consisting of Cyanine 5 and Cy5.5 (Cyanine5.5) is BHQ-3 and / or DABCYL.
  • Multiplex PCR refers to simultaneously amplifying a plurality of different target nucleic acids and / or a plurality of different amplification product species using a set of a plurality of different primers in the same reaction mixture.
  • the primers are designed so that the melting temperature (Tm) of the nucleic acid of the amplified product species is different, and a plurality of fluorescent labels capable of detecting the melting of the nucleic acid of the amplified product species are mixed in the same reaction mixture to carry out a PCR reaction.
  • Tm melting temperature
  • a plurality of fluorescent labels capable of detecting the melting of the nucleic acid of the amplified product species are mixed in the same reaction mixture to carry out a PCR reaction.
  • the differentiated-specific genomic region of the fuzarium bacterium that causes soil disease using the Abranaceae plant as a specific host is sequenced.
  • the differentiation-specific genomic region of Fuzarium bacterium which can contain the nucleotide sequence of No. 1 and causes soil diseases using lettuce as a specific host, can contain the nucleotide sequence of SEQ ID NO: 2 and is specific to spinach.
  • the differentiation-specific genomic region of Fuzarium that causes soil disease as a host can contain the nucleotide sequence of SEQ ID NO: 3, and is differentiation-specific of Fuzarium that causes soil disease using banana as a specific host.
  • the genomic region can include the nucleotide sequence of SEQ ID NO: 4.
  • a part of the nucleotide sequence unique to the differentiated fusalium strain having a brassicaceae plant as a specific host, or at least a differentiated fusalium strain having a specific host of cabbage, radish and komatsuna belonging to the brassicaceae is SEQ ID NO: It is listed as 1 in the sequence listing attached to the present specification and is shown in FIG.
  • a part of the nucleotide sequence unique to the differentiated Fusarium strain using lettuce as a specific host is listed as SEQ ID NO: 2 in the sequence listing attached herein and is shown in FIG.
  • SEQ ID NO: 3 A part of the nucleotide sequence unique to the differentiated Fusarium strain having spinach as a specific host is listed as SEQ ID NO: 3 in the sequence listing attached herein and is shown in FIG.
  • SEQ ID NO: 4 A part of the nucleotide sequence unique to the differentiated Fusarium strain having a banana as a specific host is listed as SEQ ID NO: 4 in the sequence listing attached herein and is shown in FIG.
  • Table 1 below shows the total number of nucleotides in the nucleotide sequences of SEQ ID NOs: 1 to 4 and the plants in which the differentiated Fusalium strain containing the nucleotide sequences of SEQ ID NOs: 1 to 4 is a specific host.
  • common to Brassicaceae does not mean a primer for differentiated Fuzarium bacteria whose host is all Brassicaceae plants, but refers to cabbage, daikon, komatsuna and stock of Brassicaceae. It means a primer that can detect Fuzarium bacteria having each as a specific host in common, and therefore can also detect Fuzarium bacteria having other Brassicaceae plants as specific hosts.
  • the set of primers contains an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 6, or the probe or single strand.
  • Circular templates can include the nucleotide sequence of SEQ ID NO: 5 or 6 and When the specific host is lettuce, the set of primers comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 8, or the probe or the single-stranded ring.
  • the template can contain the nucleotide sequence of SEQ ID NO: 7 or 8.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 10, or the probe or single-stranded circular template.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 12, or the probe or single-stranded circular template.
  • Table 2 below shows the nucleotide sequence of each primer of SEQ ID NOs: 5 to 12, the primer name, the number of nucleotides of the primer, and the number of nucleotides of the fragment amplified by each primer pair.
  • the set of primers contains at least two primers.
  • the set of primers can include two primers.
  • the set of primers can contain 4 to 6 primers, for example, in the LAMP method.
  • the probe can be, for example, the molecular beacon.
  • the primer set can be a pair of primers consisting of two oligonucleotides, each consisting of a sequence of 15 to 25 nucleotides in length.
  • One primer of the pair of primers contains a part of the nucleotide sequence of the differentiation-specific genomic region, and the other primer of the pair of primers contains a nucleotide sequence complementary to the nucleotide sequence of the differentiation-specific genomic region.
  • the length to the position of the nucleotide corresponding to the most 3'-terminal nucleotide of the nucleotide sequence that is identical to the complementary nucleotide sequence can be 100-500 nucleotides.
  • the nucleotide sequence of the other primer From the position of the nucleotide corresponding to the 5'-terminal nucleotide of the nucleotide sequence having the same identity as the nucleotide sequence of the one primer among the nucleotide sequences of the differentiation type-specific genomic region, the nucleotide sequence of the other primer.
  • the length to the position of the nucleotide corresponding to the most 3'-terminal nucleotide of the nucleotide sequence that is identical to the complementary nucleotide sequence is the length of the DNA fragment amplified by the two primers according to the detection method of the present invention. Equivalent to size.
  • the differentiation-type-specific genomic region of Fusarium fungi having at least two plants as specific hosts is used. It is necessary to design the primer so that the size of the contained DNA can be distinguished by the mobility of electrophoresis.
  • the GC ratio of the DNA contained in the differentiation-specific genomic region of each Fuzarium fungus should be designed so that the melting temperature does not differ significantly.
  • the set of primers in the method for detecting a host-specific differentiated Fusarium bacterium of the present invention, can be a pair of two primers.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 5 and 6.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 7 and 8.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 9 and 10 and / or.
  • the primer pair can be an oligonucleotide pair consisting of the nucleotide sequences of SEQ ID NOs: 11 and 12.
  • At least one of the primer set and the probe in the method for detecting a host-specific differentiated Fusarium bacterium of the present invention, can be labeled with a detectable atomic group.
  • the detectable atomic group is a fluorescent dye or other chromophore and one of two different molecules that specifically bind to each other by biotin, hapten, sugar chain, or other chemical or physical means. Point to.
  • biotin is the detectable atomic group of the present invention
  • the set of primers and probes labeled with biotin are easily detectable due to their very high affinity for avidin.
  • the set of primers and the probe labeled with a hapten or a sugar chain can be easily detected by an antibody or a lectin, respectively.
  • the step of detecting DNA having a nucleotide sequence contained in the differentiation-specific genomic region from a sample containing Fusalium can be carried out in a free state, such as a microplate, a DNA chip, a bead ball and the like. It can be carried out while being fixed to the solid support of.
  • a Fusarium bacterium that causes soil disease using a Fusarium family plant as a specific host and a soil using lettuce as a specific host a Fusarium bacteria that cause soil disease using a Fusarium family plant as a specific host and a soil using lettuce as a specific host.
  • Fusarium bacteria selected from the group consisting of Fusarium bacteria that cause diseases, Fusarium bacteria that cause soil diseases with spinach as a specific host, and Fusarium bacteria that cause soil diseases with banana as a specific host.
  • a step of simultaneously detecting at least two types of differentiated Fusarium bacteria having different host specificities in the same reaction mixture can be included.
  • the method for detecting a host-specific differentiated Fuzarium fungus of the present invention is a differentiation-type specific genomic region of Fuzarium fungus using a specific plant as a specific host by designing a set of primers, a probe, or a single-stranded circular template. Detection of DNA contained in is not affected by detection of DNA contained in the differentiation-specific genomic region of Fusalium fungi with any other plant as the specific host, which proceeds in the same reaction mixture. Can be done. For example, by designing primers so that different amplification product species can be distinguished by the difference in mobility due to electrophoresis, the pattern of the electrophoretic band in one lane was amplified in the same reaction mixture. It can be distinguished from other amplification product species.
  • the primers are designed so that the melting temperature (Tm) of the nucleic acid of the amplification product species is different, and a plurality of fluorescent labels capable of detecting the melting of the nucleic acid of the amplification product species are mixed and subjected to a PCR reaction to carry out the amplification product. Nucleic acid melting with temperature changes in the seed mixture can be detected by changes in fluorescence.
  • the differentiated fusalium fungi that cause soil diseases using the Abranaceae plant as a specific host are cabbage, daikon, and komatsuna.
  • Stock and other plants selected from the group consisting of Abranaceae plants can be used as specific hosts to produce differentiated fusalium fungi that cause soil diseases.
  • the sample in the method for detecting a host-specific differentiated Fusarium bacterium of the present invention, can be a soil sample.
  • the soil sample of the present invention may be any sample containing an amount of Fusarium bacteria that can infect the host. Considering the throughput, a sample that can be mixed with a primer, a probe and / or a single-stranded circular template to detect DNA having a nucleotide sequence contained in the differentiation-specific genomic region with only a minimum processing step. Is preferable.
  • a further embodiment of the present invention is a soil diagnostic method for host-specific differentiated Fusarium fungi.
  • the method for soil diagnosis of the host-specific differentiated Fusarium bacterium of the present invention uses the method for detecting the host-specific differentiated Fusarium bacterium of the present invention using the sample as a soil sample, and the host-specific differentiated Fusarium bacterium in the soil sample. And when the differentiated Fusarium fungus is detected in the soil sample by the step, it is diagnosed that the soil sample contains the differentiated Fusarium fungus, and the differentiated type in the soil sample by the step. When the Fusarium fungus is not detected, the soil sample comprises a step of diagnosing that the soil sample is free of the differentiated Fusarium fungus.
  • the method for diagnosing a host-specific differentiated Fusarium fungus of the present invention can be used to diagnose whether or not a host-specific differentiated Fusarium fungus is present in the soil of a field to be cultivated for a plant to be cultivated. ..
  • the detection limit of the soil diagnostic method for the host-specific differentiated Fusarium fungus of the present invention substantially corresponds to the minimum pathogenic fungal concentration required for growth inhibition of the host plant. Therefore, the soil diagnostic method for the host-specific differentiated Fusarium bacterium of the present invention has high sensitivity and specificity.
  • a further embodiment of the present invention is a kit for carrying out the method for detecting the host-specific differentiated Fusarium bacterium of the present invention, or a kit for carrying out the soil diagnosis method for the host-specific differentiated Fusarium bacterium of the present invention.
  • the kit of the present invention comprises a set of primers, a probe or a single-stranded circular template used in the method for detecting differentiated Fusarium fungi of the present invention and / or the method for soil diagnosis of differentiated Fusarium fungi of the present invention.
  • the differentiation-specific genomic region of Fuzarium bacterium with a specific host of Abrana family contains the DNA of the nucleotide sequence of SEQ ID NO: 1, and the differentiation-specific genomic region of Fuzarium bacterium with lettuce as a specific host is a sequence.
  • the differentiation-specific genomic region of the fuzarium bacterium containing the DNA of the nucleotide sequence of SEQ ID NO: 2 and having spinach as a specific host contains the DNA of the nucleotide sequence of SEQ ID NO: 3 and the fuzarium having a banana as a specific host.
  • the bacterial differentiation-specific genomic region contains the DNA of the nucleotide sequence of SEQ ID NO: 4.
  • the primer set is a pair of primers consisting of two oligonucleotides, each consisting of a sequence of 15 to 25 lengths.
  • One primer of the pair of primers contains a part of the nucleotide sequence of the differentiation type-specific genomic region of any one of SEQ ID NOs: 1, 2, 3 or 4, and the other primer of the pair of the primers contains the differentiation-specific specificity.
  • the nucleotide sequence complementary to the nucleotide sequence of the other primer is from the position of the nucleotide corresponding to the nucleotide at the 5'end of the nucleotide sequence of the other primer.
  • the length to the position of the nucleotide corresponding to the nucleotide at the 3'end of the nucleotide can be 100 to 500 nucleotides.
  • the set of primers contains an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 5 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 6, or the probe or single strand.
  • Circular templates can include the nucleotide sequence of SEQ ID NO: 5 or 6 and
  • the set of primers comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 7 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 8, or the probe or single-stranded circular template.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 10, or the probe or single-stranded circular template.
  • the primer set comprises an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 11 and an oligonucleotide containing the nucleotide sequence of SEQ ID NO: 12, or the probe or single-stranded circular template.
  • the set of primers, the probe, and the single-stranded circular template included in the kit of the present invention cause soil diseases using Fusarium fungi, which cause soil diseases using Abrana family plants as specific hosts, and lettuce, which causes soil diseases.
  • Differentiation of at least one Fusarium bacterium selected from the group consisting of Fusarium bacterium, Fusarium bacterium that causes soil disease using spinach as a specific host, and Fusarium bacterium that causes soil disease using banana as a specific host. Includes a portion of the nucleotide sequence of the type-specific genomic region and / or a portion of the nucleotide sequence complementary to the nucleotide sequence.
  • the set of primers, the probe and the single-stranded circular template included in the kit of the present invention further depend on the nucleic acid amplification reaction used in the step of detecting DNA having a nucleotide sequence contained in the differentiation-type specific genomic region.
  • a promoter sequence specifically recognized by T7 RNA polymerase, Nb may contain nucleotide sequences that have functions such as recognition sequences for BbvCI and other nickeling end nucleases.
  • At least one of the primer sets and probes can be labeled with a detectable atomic group.
  • Detectable atomic groups that can be used to label at least one of the primer sets and probes are 6-carboxyfluorescein (6-FAM TM), 6-carboxy-1,4-dichloro-. 2', 7'-dichlorofluorescein (TET TM), 6-carboxy-1,4-dichloro-2', 4', 5', 7'-tetrachlorofluorescein (HEX TM), VIC, NED And PET, but not limited to these.
  • Fusarium fungus that causes soil disease using the Abrana family plant as a specific host Fusarium fungus that causes soil disease using lettuce as a specific host, and spinach.
  • Fusarium fungi selected from the group consisting of Fusarium fungi that cause soil diseases with banana as a specific host and Fusarium fungi that cause soil diseases with banana as a specific host, at least two types with different host specificities. Differentiated Fusarium fungi can be detected simultaneously in the same reaction mixture.
  • At least one differentiated fusalium fungus that causes soil diseases using the Brassicaceae plant as a specific host is selected from the group consisting of cabbage, radish and komatsuna. Plants can be used as specific hosts to produce differentiated Fusalium fungi that cause soil diseases.
  • the sample in the kit of the present invention, can be a soil sample.
  • the phrase "at least N selected from the group” (N is a natural number) for a group consisting of M options includes N selected from the group and N + 1 selected from the group. It means that the number, N + 2 selected from the group, ..., M selected from the group are included. That is, the phrase “at least one selected from the group consisting of A 1 , A 2 , A 3 , ... And AM " is from “A 1 , A 2 , A 3 , ... And AM .”"One selected from the group consisting of", "A 1 , A 2 , A 3 , ...
  • the adnominal adjective "about” that modifies a numerical value means that the numerical value is in the numerical range of 90% or more and 110% or less of the numerical value.
  • “about 40 bases” refers to a base in a numerical range of 36 bases or more and 44 bases or less.
  • the differentiated Fusarium fungus used in the present specification was obtained from a research institute or a company shown as "origin" in FIG.
  • the names of 10 strains whose entire genome sequence was newly decoded in the present invention including cabbage, daikon and komatsuna, lettuce, spinach, and a differentiated fusalium strain having host specificity in bananas of the family Abrana.
  • the names of the four strains whose entire genome sequence has already been decoded used in the analysis are as shown in FIG.
  • FIG. 2 shows the estimated genome size and the size of the differentiation-type specific genomic region candidate sequence of the four strains whose entire genome sequence has already been decoded, which was used for the comparative analysis in the present invention.
  • Fuzarium bacterium is obtained by culturing 100 mL of potato dextrose liquid medium with shaking at 28 ° C. for 5 days. Add 3 mL of 2 ⁇ CTAB solution (2% cetyltrimethylammonium bromide (CTAB), 0.1M Tris-HCl, pH8.0, 1.4M NaCl, 1% polyvinylpyrrolidone) at 55 ° C. for 10 minutes. It was heated. After heating, the mixture was extracted with 3 mL of a chloroform-isoamyl alcohol (1: 1) solution, and the supernatant was further extracted 3 times with 3 mL of a chloroform-isoamyl alcohol (24: 1) solution.
  • CTAB solution 2% cetyltrimethylammonium bromide (CTAB), 0.1M Tris-HCl, pH8.0, 1.4M NaCl, 1% polyvinylpyrrolidone
  • FIG. 2 shows the estimated genome size and the size of the differentiation-type specific genomic region candidate sequence of the 10 strains newly deciphered in the entire genome sequence in the present invention, as well as the common candidate sequence for Abrana family and the common candidate sequence for Cucurbitaceae. Indicates the size of and.
  • Table 1 shows an outline of the nucleotide sequences of each Fusarium fungus differentiation-type specific genomic region obtained by the above procedure.
  • the partial sequences are listed in the sequence listing attached to the present specification as SEQ ID NOs: 1 to 4, and are shown in FIGS. 3 to 6.
  • FIG. 3 shows a part of the nucleotide sequence of a specific genomic region (SEQ ID NO: 1) common to bacteria having cabbage, radish and komatsuna as hosts of Brassicaceae.
  • Forward primer SEQ ID NO: 5
  • reverse primer common to bacteria hosting brassicaceae cabbage, radish and komatsuna (SEQ ID NO: 6) ) Is underlined.
  • FIG. 4 shows a part of the nucleotide sequence of the genomic region specific to lettuce (SEQ ID NO: 2).
  • the positions of the complementary sequences of the lettuce forward primer (SEQ ID NO: 7) and the lettuce reverse primer (SEQ ID NO: 8) are underlined.
  • FIGS. 5-1 and 5-2 show a part of the nucleotide sequence of the genomic region specific to spinach (SEQ ID NO: 3).
  • FIG. 5-1 shows the positions of the complementary sequences of the spinach forward primer (SEQ ID NO: 9) and the spinach reverse primer (SEQ ID NO: 10) underlined.
  • FIG. 6 shows a part of the nucleotide sequence of the genomic region specific to Banana (SEQ ID NO: 4).
  • the positions of the complementary sequences of the banana fungus forward primer (SEQ ID NO: 11) and the banana fungus reverse primer (SEQ ID NO: 12) are underlined.
  • PCR primer design tool Primer3 (Schgasser, A. et al., Nuclear Acid) The design was performed using Research 40: e115 (2012) and Koressaar, T. and Remm, M., Bioinformatics 23: 1289-1291 (2007)).
  • Table 2 shows the nucleotide sequence of each designed primer, the primer name, the number of nucleotides of the primer, and the number of nucleotides of the fragment amplified by each primer.
  • the position of the forward primer specific to each differentiated Fusarium fungus and the position of the complementary sequence of the reverse primer are shown by underlining the unique nucleotide sequence of each Fusarium fungus differentiated type in FIGS. 3 to 6.
  • the PCR reaction mixture was 0.5 ⁇ L of DNA sample (1 ng / ⁇ L), 1 ⁇ L of reaction buffer 10-fold concentrate (250 mM TAPS buffer (pH 9.3, 25 ° C.), 500 mM KCl, 20 mM MgCl 2 , 1 mM DTT), 0.8 ⁇ L deoxyribonucleoside mixture (2.5 mM dNTP mixture, respectively), 1 ⁇ L forward primer solution (10 ⁇ M), 1 ⁇ L reverse primer solution (10 ⁇ M), 0.05 ⁇ L Extaq (registration). Trademark) An enzyme solution (5 U / ⁇ L) and 5.65 ⁇ L of pure water were mixed to make 10 ⁇ L.
  • reaction conditions were as follows: after 30 seconds at 95 ° C., each cycle was set to 95 ° C. for 15 seconds, 60 ° C. for 30 seconds, 72 ° C. for 30 seconds, and this was repeated for 30 cycles, and then heated at 72 ° C. for 5 minutes. After that, it was stored refrigerated at 4 ° C. After the PCR reaction, the DNA was electrophoresed on a 1% agarose gel, stained with 1 ⁇ g / mL ethidium bromide, and imaged under a UV illuminator.
  • FIG. 7 is a panel of an electrophoretogram showing the detection of Fusarium fungi by the PCR method using the common differentiation-specific primer pair of Brassicaceae and the common primer pair of Fusarium fungi of the present invention.
  • the electrophoretogram on the panel shows differentiated Fuzarium fungi hosting each of Brassicaceae cabbage, daikon and komatsuna, and differentiated Fuzarium fungi hosting other Brassicaceae plants (Stock fungus (1) and (2)), Banana fungus, Cucumber fungus, and non-pathogenic Fuzarium fungus samples were subjected to PCR reaction using a pair of differentiation-type specific primers common to Brassicaceae fungi. Shows an electrophoresis band.
  • the electrophoretogram below the panel shows the Fuzarium fungus extracellular matrix protein 1 (FoFEM1) constitutively expressed in all Fuzarium fungi reported by Michiels et al. (LOS Pathog 5 (10): e10000637 (2009)) as a positive control.
  • FoFEM1 Fuzarium fungus extracellular matrix protein 1
  • a pair of gene-specific primers is a differentiated fusalium fungus whose host is cabbage, daikon, and komatsuna belonging to the abrana family, and a differentiated fuzalium fungus whose host is another abrana family plant (stock fungus (1) and (2)), agarose gel electrophoresis band of the reaction product obtained as a result of carrying out a PCR reaction using a sample of banana fungus, cucumber fungus, and non-pathogenic fusalium fungus is shown.
  • the "stock bacteria (1)” and "stock bacteria (2)” are referred to as Fusarium oxysporum f. Sp. Two strains with different matthiolae (880129h and 851209g, respectively).
  • the Brassicaceae common primer pair amplified a large amount of 217 bp DNA fragments as designed from the Brassicaceae sample.
  • the Brassicaceae common primer pair amplified DNA of the same size from samples of one strain each of Banana and Cucumber.
  • the amount of DNA amplified from the strain samples of Banana and Cucumber was significantly smaller than that amplified from the sample of Fusarium fungus whose host was Brassicaceae. This is because the banana and cucumber strains contain nucleotide sequences that are slightly homologous to the differentiated-specific genomic regions of cabbage, daikon, and komatsuna, but the homology with the primer sequences is low, so the amount is very small.
  • FIG. 8 is a table showing the results of a detection test of a Fusarium strain by the PCR method using the above-mentioned four types of primer pairs for the Fusarium strain differentiated type of 146 strains.
  • Each column of the table in FIG. 8 contains a differentiation-specific primer pair for spinach, lettuce, and banana, a differentiation-specific primer pair common to Brassicaceae, and a primer pair common to Fusarium, and each primer pair.
  • the specific detection accuracy of the detection test used is shown. The specific detection accuracy is expressed as a percentage of the number of strains obtained by subtracting the number of false-negative and false-positive differentiated strains from the total number of differentiated strains tested (146 strains).
  • Each row in the table shows the sample Fusarium strains (lettuce, onion, spinach, pea, other legumes, bananas, tomatoes, cucumbers, melons, other cucurbitaceae, cabbage, etc. Shows cucurbita, lettuce, stock and non-pathogenic bacteria).
  • the denominator of the fractional notation in the table of FIG. 8 indicates the number of differentiated Fusarium strains of the sample in each row, and the numerator indicates the number of differentiated Fusarium strains that could be accurately detected.
  • the differentiation-specific primer pairs of spinach, lettuce, and banana bacteria detected only each differentiated type of Fusarium. Therefore, the specific detection accuracy of the differentiation-specific primer pair of spinach, lettuce, and banana and the foFEM1 gene-specific primer pair was 100%.
  • the differentiation-specific primer pair common to Brassicaceae also reacts with two Brassicaceae (stock bacteria) strains other than cabbage, radish, and komatsuna used to determine the unique genome sequence specific to differentiation. However, a reaction was observed with each of the banana and cucumber strains. Therefore, the specific detection accuracy of the differentiation-specific primer pair common to Brassicaceae was 98.6%. As shown in FIG.
  • the amount of DNA amplified from the strain samples of Banana and Cucumber was significantly smaller than that of the DNA amplified from the Brassicaceae sample. This is because the banana and cucumber strains contain nucleotide sequences that are slightly homologous to the differentiation-specific genomic regions of cabbage, radish, komatsuna, and stock, but have low homology with primer sequences. Therefore, it is considered that a small amount of DNA was amplified.
  • soil diagnosis of a host-specific differentiated Fusarium bacterium is performed using the method for detecting a host-specific differentiated Fusarium bacterium of the present invention, it is easy to make a true positive and a false positive because the difference in the amount of amplified DNA is large. It does not matter in practice because it can be identified as.
  • FIG. 9 shows the results of multiplex PCR reaction on a mixed sample of cabbage, spinach, lettuce and banana using a primer pair specific to cabbage, spinach, lettuce and banana of the present invention. It is an electrophoretogram which shows. “Cabbage”, “spinach”, “lettuce” and “banana” in FIG. 9 represent lanes of PCR reaction products using primer pairs and cell samples specific to each, and “Mixture (4). Strain) ”represents a lane of PCR reaction products using four differentiated specific primer pairs and cell samples. As shown by the band of the lane of "Mixture (4 strains)" in FIG. 9, it was proved that the primer pair of the present invention can simultaneously detect multiple types of Fusarium bacteria in soil by the multiplex PCR method.
  • FIG. 10 shows Fusarium fungi from soil samples of artificially contaminated soil by the PCR method using the differentiation-specific common primer pair of Abrana family fungus, the differentiation-specific primer pair of Fusarium fungus, or the common primer pair of Fusarium fungus of the present invention. It is a panel of an electrophoretic diagram showing the detection of.
  • the electrophoretogram on the panel of FIG. 10 was obtained as a result of performing a PCR reaction using a soil sample of artificially contaminated soil supplemented with onion, cabbage and spinach and a pair of common primers for Brassicaceae.
  • the agarose gel electrophoresis band of the reaction product is shown.
  • FIG. 10 shows the results of PCR reaction using a soil sample of artificially contaminated soil supplemented with onion, cabbage or spinach and a pair of differentiation-specific primers of spinach.
  • the agarose gel electrophoresis band of the obtained reaction product is shown.
  • the electrophoretic diagram at the bottom of the panel of FIG. 10 shows the reaction obtained as a result of performing a PCR reaction using a soil sample of artificially contaminated soil supplemented with onion fungus, cabbage fungus or spinach fungus and a pair of Fuzarium fungus common primers.
  • the agarose gel electrophoresis band of the product is shown.
  • the Brassicaceae common primer amplified DNA from soil samples containing cabbage, but did not amplify DNA from soil samples containing other bacteria.
  • the spinach primer amplified DNA from soil samples containing spinach, but not from soil samples containing other bacteria.
  • the FoFEM1 gene-specific primer pair amplified DNA from all soil samples containing Fusarium. From the results of FIG. 10, it was proved that the method for detecting host-specific differentiated Fusarium fungi of the present invention can detect host-specific differentiated Fusarium fungi from soil samples. Therefore, it was proved that the soil diagnosis method for the host-specific differentiated Fusarium fungus using the method for detecting the host-specific differentiated Fusarium fungus of the present invention is also feasible.
  • FIG. 11 is an electrophoretogram showing the detection sensitivity of spinach in soil using the spinach primer pair of the present invention.
  • the reaction product obtained by performing a PCR reaction using a part of the soil inoculated with 10 to 106 spinach spores per 1 g of soil as a sample was detected by agarose gel electrophoresis.
  • "Mock” represents the lane of the PCR reaction product of the soil sample to which the spore was not added
  • " 101 " to " 106 " represent the lane of the PCR reaction product of the soil sample having the spore concentration.
  • M represents a lane of standard polynucleotide markers comprising 100 bp, 200 bp and 300 bp.
  • the DNA fragment of the reaction product was amplified in the PCR reaction using the soil sample inoculated with 102 to 106 pieces per 1 g of soil and the spinach primer pair.
  • the DNA fragment of the reaction product could not be amplified by the PCR reaction using the soil sample inoculated with 10 pieces per 1 g of soil, the soil sample to which the spores were not added (Mock), and the spinach primer pair. Therefore, it was proved that the detection limit of the soil diagnostic method for the host-specific differentiated Fusarium bacterium of the present invention is 102 per gram for spinach bacterium.
  • FIG. 12 is a photograph of the results of an inoculation experiment showing the relationship between the concentration of inoculated cells of the spinach bacterium of the present invention and the growth inhibitory effect of the host spinach.
  • “Mock” represents the soil not inoculated with Fusarium spores
  • “ 101" to “10 4 " represents the soil inoculated with the spore concentration of spinach.
  • the detection limit of the method for detecting the host-specific differentiated Fusarium fungus of the present invention and the minimum concentration of Fusarium fungi required for growth inhibition of the host plant are both 1 g. There were 102 per. Therefore, it was shown that the soil diagnostic method for host-specific differentiated Fusarium bacteria of the present invention can detect Fusarium bacteria having the minimum onset concentration, and thus has sufficient detection sensitivity for practical use.
  • the host specificity of the differentiated type of Fusarium fungus has only been evaluated by conducting an infection test to see if each plant causes soil disease.
  • all of the methods for detecting host-specific differentiated Fusarium fungi of the present invention were able to detect host-specific differentiated Fusarium fungi with a detection accuracy of at least 98.6% or more. Infection tests can only be performed during the period in which the plant is cultivated, and it takes time for signs of disease to be observed.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention is excellent in that results can be obtained in a short time whenever a Fusarium bacterium cell is present.
  • the method for detecting a host-specific differentiated Fusarium bacterium of the present invention has sufficient detection sensitivity for practical use.
  • the method for diagnosing soil of a host-specific differentiated Fusarium bacterium of the present invention has similar advantages. Further, the soil diagnosis method of the present invention has sufficient detection sensitivity for practical use. Therefore, it can be said that the present invention has industrial applicability. It was

Abstract

La présente invention concerne un procédé de détection d'une forme spécifique de l'hôte de Fusarium oxysporum, ledit procédé comprenant une étape de détection, à partir d'un échantillon contenant F. oxysporum, d'ADN ayant une séquence nucléotidique contenue dans la région génomique spécifique de la forme spécifique d'au moins une souche de F. oxysporum, choisie dans le groupe constitué par F. oxysporum, etc. qui utilise les plantes Brassica comme hôte spécifique et provoque des maladies du sol, avec l'utilisation d'une paire d'amorces, d'une sonde ou d'un modèle cyclique simple brin ayant une partie de la souche susmentionnée et une partie d'une séquence nucléotidique qui est complémentaire de la séquence nucléotidique susmentionnée.
PCT/JP2021/041612 2020-11-11 2021-11-11 Procédé de diagnostic du sol fusarium oxysporum par génomique du pathogène WO2022102726A1 (fr)

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