WO1994019489A2 - Eyespot dna-sequence - Google Patents
Eyespot dna-sequence Download PDFInfo
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- WO1994019489A2 WO1994019489A2 PCT/EP1994/000516 EP9400516W WO9419489A2 WO 1994019489 A2 WO1994019489 A2 WO 1994019489A2 EP 9400516 W EP9400516 W EP 9400516W WO 9419489 A2 WO9419489 A2 WO 9419489A2
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- Prior art keywords
- eyespot
- septoria
- fusarium
- dna
- probe
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- C—CHEMISTRY; METALLURGY
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- 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
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- 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
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- 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
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to a DNA-Sequence which is useful as a probe specific for eyespot.
- the growth, development and yield of a plant depends on its health and this in its turn can be affected by phytopathogenic fungi.
- One of the most serious fungi to affect cereals is that known as eyespot (Pse ⁇ docercosporel/a herpotrichoides). This organism causes a lesion in the stem of the plant thus weakening it and results in a condition known as "lodging" whereby the crop collapses giving rise to substantial yield losses.
- the disease can inhibit the growth of the plant.
- an oligonucleotide sequence preferably a sequence which contains 1 6 to 20 nucleotides, such as the following sequence:
- TCAAGGAAGAAACTCGTC 3' can be used as a probe specific for eyespot which does not hybridize with other phytopathogenic fungi, such as Fusarium, Septoria, Rhizoctonia or with cereals such as wheat itself.
- the probe can form the basis of a diagnostic kit for the in vivo detection of eyespot infection in plants.
- ribosomal RNA allows the development of probes capable of detecting and identifying a small number of microorganisms in clinical isolates or in complex natural populations.
- Ribosomal RNA is composed of conserved and partially non-conserved regions. Species specific sequences can be found in these partially non-conserved regions by comparison of the sequence of the target ribosomal gene with ribosomal RNA sequences of related species.
- oligonucleotide probes can be synthesized and tested for specificity, selectivity and efficacy.
- an oligonucleotide sequence preferably a sequence, which contains 25 to 30 nucleotides, such as the following sequence:
- 5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' can be used as an amplification primer for hybridisation of the DNA-strains of Fusarium, Septoria and eyespot.
- RNA ⁇ enes of evesoot Fusarium and Septoria
- the amplification by PCR of DNA from eyespot, Fusarium and Septoria was obtained according to the protocol supplied by Perkin-Elmer Cetus.
- Oligonucleotide primers were the same as those used by Medlin et. al. (Gene, 71 (1988) 491-499: "The characterization of enzymatically amplified eukaryotic 1 6S-like rRNA coding regions"). These oligonucleotides contained restriction sites that can be used for subcloning.
- the conditions used for amplification were: 200 ng of DNA
- the amplification was:
- Eyespot ribosomal DNA was shown to contain an EcoRI site located 1 .6 kb from the 5' end of the 1 .9 kb PCR products. Septoria and Fusarium do not contain this site. Mapping of the site along the ribosomal gene was performed by Southern blot analysis (Amersham protocol), using probes P1 or P2 labelled with 30mCi of y 32 ? ATP (3000Ci/mmol).
- the conditions of the hybridization were 1 hour at 50 °C in a buffer (5x SSC (NaCI and sodium citrate), 5x Denhardt buffer and 0.5% SDS (sodium dodecyl sulfate)). The blot was washed for 1 hour at 65 °C in 0.5 SSC buffer (see Fig. 2).
- Cloning insertion of cleavage products into the M13mp1 8 vector DNA amplified from eyespot, Fusarium and Septoria were purified and digested by EcoRI and Hindlll. The cleavage products were purified by gel electrophoresis.
- Septoria and Fusarium DNAs were inserted into bacteriophage vector M13mp18 that were cut by EcoRI-Hindlll (10U/ /g DNA of each enzyme at 37 °C during 1 -2 hours).
- the two eyespot DNA fragments were cloned into either the EcoRI site (1 .6 kb fragment) or the EcoRI-Hindlll (0.3kb fragment) site of M1 3mp1 8.
- Positive clones were detected by filter hybridization of the replica plates with P1 or P2 oligonucleotides as probes, according to the protocol supplied by Amersham.
- DNAs from the positive clones were isolated, cleaved by EcoRI and Hindlll, analysed by gel electrophoresis (1 % Agarose gel with 0.5 ⁇ g/ml of ethidium bromide) and Southern blot hybridization as described in Example 2 (see Fig. 3).
- Ml 3 primers were used to initiate sequencing from the Hindlll cloning site. Internal specific primers were used to sequence 1 kb around the EcoRI site of eyespot.
- Both strands of the DNA were sequenced with the universal M13 primer and the reverse M1 3 primer.
- EYESPOT GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAAACCTTGTTACG 55
- EYESPOT GTTGCCACCCTCTCTGGGCCGGTCCAAAGGCCTCACTTAGCCATTCAATCGGTAG 165
- Septoria GTTGCCGACCTCCCTG AGCCAGTCCGAAGGCCTCACTGAGCCATTCAATCGGTAG 165
- Fusarium GTTGCCCACCTCTCTGGGCCAGTCCGGACGCCTCACTGAGCCATTCAATCGGTAG 165
- EYESPOT TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAGCGCAAGCTGATGA 219 Septoria: TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAACGCATGCTGATGA 21 9 Fusarium: TAGCGACGGGCGGTGTGTACAAAGGGNAGNGTACGTAATCAACGCAAGCTGATGA 220
- EYESPOT CTTGCGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 t I
- EYESPOT CAGCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA-AACT 328
- EYESPOT CGTC.GGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAGGGCATCAC 383 Septoria: CGTTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 383 Fusarium: CGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 382
- EYESPOT AGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Septoria: AGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Fusarium: AGACCTGTTATTGCCTCAAACTTCCATCGGNTTGAGCCGATAGTCCCTCTAAGAA 437
- EYESPOT GCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493
- EYESPOT ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTGTGTCTGGACC 603
- Septoria ACCCACAAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTACTTGTGTCTGGACC 603
- Fusarium ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCATTGTGTCTGGACC 602
- EYESPOT GNCCTTCCGTCAATTTCTTTAAGTTTCAGNCTTGGGACCATACTCCCCCCAGTAA 713 Septoria: GNCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCATACTCCCCCCAG-AA 712 Fusarium: GCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCNTACTCCCCCTGGAGN 712
- EYESPOT CCCAAAGACTTTGNTTTCTCGTAAGGTGCCGAGCGAGTCAAAAAAATAACATTGC 768 Septoria: CCCAAAAACTTTGNTTTCTGGTAAGGTGCCGATGGAGTCAAAAAGATAACATCCA 767 Fusarium: CC-AAGCACTTTGNTTTCTCGTAAGGTGCCGAACGGGTCAAAAAA-TAACACCGT 765
- EYESPOT CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 823
- EYESPOT TCGATCCCCTAACTTTCGGTCACTGTATTAATGAAAACATCCTTGGCAAATGCTT 878
- EYESPOT TCGCAGNAGTTAGTCTTCAATAAATCCAAGTAATTTCACCTCTGACAATTGTAAT 933
- the sequences were analyzed by using PC-Gene software. Alignment of sequences reveals a high level of homology between eyespot, Fusarium and Septoria DNAs. Analysis of restriction sites confirms that an EcoRI site is specifically present in eyespot DNA at position 240 and reveals other restriction polymorphisms. An Nhel site was found exclusively in eyespot at position 456.
- an eyespot specific probe of 18 nt long fragment has been defined which is located between positions 31 5 and 332.
- This probe contains 7 nucleotides different from the sequences of Fusarium and Septoria.
- Sequence data were also used to define a new amplification primer able to hybridize with the DNA of the three strains analyzed.
- This primer referred to as P6, allows the amplification of a 940 nt long fragment.
- Amplifications were performed by using DNA of the original eyespot, on the DNA of Septoria, Fusarium, and by using DNA obtained from 27 various strains of eyespot (see Fig. 5).
- primers P2 and P6 allow an amplification of all the DNA tested and gives a fragment of 940 bp.
- eyespot strain WF61 1 (referred to as number 3 in Fig. 5) gives a different pattern, with an amplified fragment of 1.2 kb, probably resulting from an insertion.
- Example 1 The protocol used for the amplification of various eyespot strains is described in Example 1 .
- Amplified DNAs were subjected to restriction by EcoRI and Nhel (EcoRI (10U/mg) and Nhel (10U/ ⁇ g) over 1 -2 hours at 37°C).
- the EcoRI site is found in all eyespot strains analyzed with the exception of WUK182 (referred to as number 10 in the Fig. 5)
- the Nhel site is present in all eyespot strains.
- DNA from various eyespot strains and from Fusarium and Septoria were amplified as described in Example 4, and subjected to Southern blot analysis. Hybridization was performed with radiolabelled EP1 eyespot probe.
- the EP1 probe hybridizes with all the eyespot strains, with the exception of strain WUK182 (referred to as number 10 in Fig. 6).
- the eyespot probe does not hybridize with Septoria or Fusarium DNA.
- Hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer) with radiolabelled EP1 probe. The washes were at 75 °C over 30 mins in a 0.1 SSC buffer.
- Sal I purified and cloned into the M13mp18 vector, as described in Example 3, in order to analyse its sequence.
- Alignment of the sequences of WUK182, Septoria, Fusarium and the original eyespot shows that: a) the EcoRI site is not present in WUK182 DNA, due to a transition A to C, b) at the level of the probe, WUK182 sequence differs from the original eyespot sequence by 5 bp.
- EYESPOT TTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50 Septoria TTACTAGGCATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50
- Amplifications and hybridizations were performed on DNA extracted from wheat, eyespot infected wheat, and Rhizoctonia cerealis. Amplification products were run on gels and southern blot was hybridized with the EP1 eyespot probe (see Fig. 7).
- the hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer).
- the EP1 probe hybridized to infected wheat, eyespot, but not with Rhizoctonia cerealis nor with Septoria or Fusarium, as noted above.
- EP1 is a probe specific for eyespot which does not hybridize with Fusarium, Septoria, Rhizoctonia, nor with wheat itself. In terms of specificity, and considering the small number of various species analyzed, this probe can be considered as 100% specific. In terms of range of selectivity, and considering the 28 different strains tested, the probe can be considered as 96.5 % selective.
- Fig. 1 shows the gel after the amplification of ribosomal RNA genes of eyespot, Fusarium and Septoria.
- the samples were loaded onto a 0.8 % agarose gel and electrophoretically separated.
- the DNA was visualized by ethidium bromide (0.5 mg/ml in agarose gel) under U.V.
- the marker of the DNA is I (lambda) DNA digested by EcoRI and Hindlll.
- Fig. 2 shows the EcoRI I Hindlll cleavage sites on the 1.9 kb long DNA-fragment with the position of the two primers (P1 and P2).
- Fig. 3 shows the Southern blot of miniprep DNAs (from the positive clones) hybridized with probe P2 and probe P1 .
- Probe P2 is specific for the 0.3 kb EcoRI-Hindlll fragment of Eyespot and the 2 kb EcoRI-Hindlll fragment of Fusarium and Septoria.
- Probe P1 is specific for the 1 .6 kb EcoRI fragment of eyespot.
- Fig. 4 shows the respective positions of primers P2 and P6, EcoRI and Nhel restriction sites and EP1 probe.
- Fig. 5 shows the gel after the amplification of various strains of eyespot, Fusarium and Septoria DNA using both Primers 2 and 6.
- the samples were electrophoretically separated on a 1 % agarose gel, and visualized by ethidium bromide (0.5 ⁇ g/ml in the agarose gel).
- the marker is ⁇ (lambda) DNA digested by EcoRI-Hindlll.
- Fig.6 shows the results of the hybridization of the amplified products with the eyespot probe, EP1.
- Fig.7 shows the analysis of the hybridization in plants.
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Abstract
Oligonucleotide sequences which can be used as a probe specific for eyespot which do not hybridize with fungi such as Fusarium, Septoria, Rhizoctonia or with cereals such as wheat itself, as well as oligonucleotide sequences which can be used as amplification primers for hybridisation of the DNA-strains of Fusarium, Septoria and eyespot are described. The probe can form the basis of a diagnostic kit for the in vivo detection of eyespot infection in plants.
Description
Title: Evespot DNA-seαuence
Field of the invention
The present invention relates to a DNA-Sequence which is useful as a probe specific for eyespot.
The growth, development and yield of a plant depends on its health and this in its turn can be affected by phytopathogenic fungi. One of the most serious fungi to affect cereals is that known as eyespot (Pseυdocercosporel/a herpotrichoides). This organism causes a lesion in the stem of the plant thus weakening it and results in a condition known as "lodging" whereby the crop collapses giving rise to substantial yield losses. The disease can inhibit the growth of the plant. There are fungicides available to combat this organism, but treatment has to be carried out before the symptoms of the attack are visible. Thus, a method of detecting the early presence of this fungus in plants is highly desirable.
It has now been found that an oligonucleotide sequence, preferably a sequence which contains 1 6 to 20 nucleotides, such as the following sequence:
5' TCAAGGAAGAAACTCGTC 3' can be used as a probe specific for eyespot which does not hybridize with other phytopathogenic fungi, such as Fusarium, Septoria, Rhizoctonia or with cereals such as wheat itself.
The probe can form the basis of a diagnostic kit for the in vivo detection of eyespot infection in plants.
Knowledge of ribosomal RNA allows the development of probes capable of detecting and identifying a small number of microorganisms in clinical isolates or in complex natural populations.
Ribosomal RNA is composed of conserved and partially non-conserved regions.
Species specific sequences can be found in these partially non-conserved regions by comparison of the sequence of the target ribosomal gene with ribosomal RNA sequences of related species.
After sequencing, alignment of sequences and determination of variable spots, oligonucleotide probes can be synthesized and tested for specificity, selectivity and efficacy.
It has further been found that an oligonucleotide sequence, preferably a sequence, which contains 25 to 30 nucleotides, such as the following sequence:
5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' can be used as an amplification primer for hybridisation of the DNA-strains of Fusarium, Septoria and eyespot.
The following Examples describe the identification and isolation of the DNA-sequences of the oligonucleotides of the invention.
Example 1
Amplification of ribosomal RNA αenes of evesoot. Fusarium and Septoria The amplification by PCR of DNA from eyespot, Fusarium and Septoria was obtained according to the protocol supplied by Perkin-Elmer Cetus.
Oligonucleotide primers were the same as those used by Medlin et. al. (Gene, 71 (1988) 491-499: "The characterization of enzymatically amplified eukaryotic 1 6S-like rRNA coding regions"). These oligonucleotides contained restriction sites that can be used for subcloning.
_ EcoRI-x
P1 5' GGG CCG AAT TCG TCG ACA ACC TGG GGT AGC CTG CCA GT 3'
P2 5' GCC CGG GAT CC :,AA AAGGCC TTTT..GC ATC CTT CTG CAG GTT CAC CTA C 3' *- Hind ///•-
The conditions used for amplification were:
200 ng of DNA
2.4 pmol of each primer (P1 and P2) Manufacturer's supplied buffer (Cetus) dNTPs (200mM each) Taq DNA poiymerase at 25U/ml (Perkin Elmer Cetus).
The amplification was:
Initial denaturation process 5' at 94°C, and 30 cycles of 2' at 94°C, 1 ' at 55 °C and 4' at 72°C.
All molecular biological manipulations were done according to Maniatis T, Fritsch E F, and Sambrook J, (1 982) Molecular Cloning, a Laboratory Manual); (see also Fig. 1 ).
Example 2 Evidence for an EcoRI polymorphism
Eyespot ribosomal DNA was shown to contain an EcoRI site located 1 .6 kb from the 5' end of the 1 .9 kb PCR products. Septoria and Fusarium do not contain this site. Mapping of the site along the ribosomal gene was performed by Southern blot analysis (Amersham protocol), using probes P1 or P2 labelled with 30mCi of y32? ATP (3000Ci/mmol).
The conditions of the hybridization were 1 hour at 50 °C in a buffer (5x SSC (NaCI and sodium citrate), 5x Denhardt buffer and 0.5% SDS (sodium dodecyl sulfate)). The blot was washed for 1 hour at 65 °C in 0.5 SSC buffer (see Fig. 2).
Example 3
Cloning: insertion of cleavage products into the M13mp1 8 vector DNA amplified from eyespot, Fusarium and Septoria were purified and digested by EcoRI and Hindlll. The cleavage products were purified by gel electrophoresis.
Septoria and Fusarium DNAs were inserted into bacteriophage vector M13mp18 that were cut by EcoRI-Hindlll (10U/ /g DNA of each enzyme at 37 °C during 1 -2 hours).
The two eyespot DNA fragments were cloned into either the EcoRI site (1 .6 kb fragment) or the EcoRI-Hindlll (0.3kb fragment) site of M1 3mp1 8.
Example 4 Screening of positive clones
Positive clones were detected by filter hybridization of the replica plates with P1 or P2 oligonucleotides as probes, according to the protocol supplied by Amersham.
Example 5
DNA analysis of positive clones
The DNAs from the positive clones were isolated, cleaved by EcoRI and Hindlll, analysed by gel electrophoresis (1 % Agarose gel with 0.5 μg/ml of ethidium bromide) and Southern blot hybridization as described in Example 2 (see Fig. 3).
Example 6
Seouence analysis
Sequence analysis was performed using an automatic DNA sequencer. Universal
Ml 3 primers were used to initiate sequencing from the Hindlll cloning site. Internal specific primers were used to sequence 1 kb around the EcoRI site of eyespot.
Both strands of the DNA were sequenced with the universal M13 primer and the reverse M1 3 primer.
The sequence surrounding the EcoRI site of eyespot is shown below along with the equivalent region of Fusarium and Septoria:
EYESPOT: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAAACCTTGTTACG 55 Septoria: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAGAACTTGTTACG 55
Fusarium: GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAGACCTTGTTACG 55
EYESPOT: ACTTTTACTTCCTCTAAATGACCAAGTTTGTACGATTTTCCAGCCCTGAGTGGGT 1 10 Septoria: ACTTTTACTTCCTCTAAATGACCGAGTTTGACCAACTTTCCGGCTCTGGGTGGTC 1 10 Fusarium: ACTTTTACTTCCTCTAAATGACCGAGTTTGGAGAGCTTTCCGGNCCTGAGTGGTA 1 10
EYESPOT: GTTGCCACCCTCTCTGGGCCGGTCCAAAGGCCTCACTTAGCCATTCAATCGGTAG 165 Septoria: GTTGCCGACCTCCCTG AGCCAGTCCGAAGGCCTCACTGAGCCATTCAATCGGTAG 165 Fusarium: GTTGCCCACCTCTCTGGGCCAGTCCGGACGCCTCACTGAGCCATTCAATCGGTAG 165
EYESPOT: TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAGCGCAAGCTGATGA 219 Septoria: TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAACGCATGCTGATGA 21 9 Fusarium: TAGCGACGGGCGGTGTGTACAAAGGGNAGNGTACGTAATCAACGCAAGCTGATGA 220
r ECORI .
EYESPOT: CTTGCGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 t I
Septoria: CATGCGCTTACTAGGCATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274 Fusarium: NTTGNGCTTACTAGGGATTCCTCGTTGANGAGCAATAATTGCAATGNTCTATCCC 275
j EP1 Probe —
EYESPOT: CAGCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA-AACT 328
Septoria: CAGCACGACGGAGTTTAACAAGATTACCCAGACCTTTCGGCCAAGGTGATGAACT 329 Fusarium: CAGCACGANGGNGTTTNTCAAGNATACCCGGACCTTTCGGACAAGGAA--GTACT 328
EYESPOT: CGTC.GGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAGGGCATCAC 383 Septoria: CGTTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 383 Fusarium: CGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAGGGCATCAC 382
EYESPOT: AGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Septoria: AGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438 Fusarium: AGACCTGTTATTGCCTCAAACTTCCATCGGNTTGAGCCGATAGTCCCTCTAAGAA 437
_ VΛe/_
I '
EYESPOT: GCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493
Septoria: GCCGGCGACCCGCCAAAGCGGGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493 Fusarium: GCCAGCGTACTGCCAAAGCAATACGGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 492
EYESPOT: TATCGCAATTAAGCAGACAANTCACTCCACCAACTAAGAACGGTCATGCACCACC 548 Septoria: TATCGCAATTAAGCAGACAAATCACTCCACCAACTAAGAACGGTCATGNACCACC 548 Fusarium: TATCGCAATTAAGCAGACAAATCACTCCACCAACTAAGAACGGCCATGCACCACC 547
EYESPOT: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTGTGTCTGGACC 603 Septoria: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTACTTGTGTCTGGACC 603 Fusarium: ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTCATTGTGTCTGGACC 602
EYESPOT: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGCGGT 658 Septoria: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACGCCTGGTGGT 658 Fusarium: TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACCCCTGGTGGT 657
EYESPOT: GNCCTTCCGTCAATTTCTTTAAGTTTCAGNCTTGGGACCATACTCCCCCCAGTAA 713 Septoria: GNCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCATACTCCCCCCAG-AA 712 Fusarium: GCCCTTCCGTCAATTTCTTTAAGTTTCAGCCTTGGGACCNTACTCCCCCTGGAGN 712
EYESPOT: CCCAAAGACTTTGNTTTCTCGTAAGGTGCCGAGCGAGTCAAAAAAATAACATTGC 768 Septoria: CCCAAAAACTTTGNTTTCTGGTAAGGTGCCGATGGAGTCAAAAAGATAACATCCA 767 Fusarium: CC-AAGCACTTTGNTTTCTCGTAAGGTGCCGAACGGGTCAAAAAA-TAACACCGT 765
EYESPOT: CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 823
Septoria: CCGATCCCTAGTCGGNATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 822 Fusarium: CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 820
EYESPOT: TCGATCCCCTAACTTTCGGTCACTGTATTAATGAAAACATCCTTGGCAAATGCTT 878
Septoria: TCGATCCCCTAACTTTCGTTCACTG-ATTAATGAAAACATCCTTGGCAAATGCTT 876 Fusarium: TCGATCCCCTAACTTTCGTTC-CTG-ATTAATGAAAACATCCTTGGCAAATGCTT 873
EYESPOT: TCGCAGNAGTTAGTCTTCAATAAATCCAAGTAATTTCACCTCTGACAATTGTAAT 933
Septoria: TCGNAGTAGTTCGTCTTCAATAAATCCAAG-AATTTCACCTCTGACAATTG-AAT 929 Fusarium: TCGNAGNAGTTAGTCTTCAATAAATCCAAG-AATTTCACCTCTGACAATTG-AAT 926
N = not known - = missing
The sequences were analyzed by using PC-Gene software. Alignment of sequences reveals a high level of homology between eyespot, Fusarium and Septoria DNAs. Analysis of restriction sites confirms that an EcoRI site is specifically present in eyespot DNA at position 240 and reveals other restriction polymorphisms. An Nhel site was found exclusively in eyespot at position 456.
From the alignment of the sequences, an eyespot specific probe of 18 nt long fragment has been defined which is located between positions 31 5 and 332. This probe contains 7 nucleotides different from the sequences of Fusarium and Septoria.
Sequence of the probe: 5' TCAAGGAAGAAACTCGTC 3' This probe will be referred to as EP1.
Sequence data were also used to define a new amplification primer able to hybridize with the DNA of the three strains analyzed. This primer, referred to as P6, allows the amplification of a 940 nt long fragment.
Sequence of the primer P6: 5' GCCGTCGACCAATTGTCAGAGGTGAAA 3' The respective positions of the primers and probe are shown in Fig. 4.
Example 7
Control of the specificity of the probe
a) Amplification of various eyespot strains
Amplifications were performed by using DNA of the original eyespot, on the DNA of Septoria, Fusarium, and by using DNA obtained from 27 various strains of eyespot (see Fig. 5).
The combination of primers P2 and P6 allows an amplification of all the DNA tested and gives a fragment of 940 bp. However, eyespot strain WF61 1 (referred to as number 3 in Fig. 5) gives a different pattern, with an amplified fragment of
1.2 kb, probably resulting from an insertion.
The protocol used for the amplification of various eyespot strains is described in Example 1 .
Example 8
Restriction enzyme analysis
Amplified DNAs were subjected to restriction by EcoRI and Nhel (EcoRI (10U/mg) and Nhel (10U/μg) over 1 -2 hours at 37°C).
The EcoRI site is found in all eyespot strains analyzed with the exception of WUK182 (referred to as number 10 in the Fig. 5)
The Nhel site is present in all eyespot strains.
Restriction enzyme analysis confirms that eyespot WF61 1 rDNA contains an insertion.
Example 9 Hybridization of the amplified products with the evesoot probe
DNA from various eyespot strains and from Fusarium and Septoria were amplified as described in Example 4, and subjected to Southern blot analysis. Hybridization was performed with radiolabelled EP1 eyespot probe.
The results are shown in Fig. 6.
The EP1 probe hybridizes with all the eyespot strains, with the exception of strain WUK182 (referred to as number 10 in Fig. 6). The eyespot probe does not hybridize with Septoria or Fusarium DNA.
Hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer) with radiolabelled EP1 probe. The washes were at 75 °C over 30 mins in a 0.1 SSC buffer.
Example 10
Seouence analysis of evespot strain WUK1 82
Amplified DNA from WUK182 (clone No. 10 in Fig. 6) was digested by Hindlll and
Sal I, purified and cloned into the M13mp18 vector, as described in Example 3, in order to analyse its sequence.
Alignment of the sequences of WUK182, Septoria, Fusarium and the original eyespot shows that: a) the EcoRI site is not present in WUK182 DNA, due to a transition A to C, b) at the level of the probe, WUK182 sequence differs from the original eyespot sequence by 5 bp.
r EcoRI—j
EYESPOT TTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50 Septoria TTACTAGGCATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCCCA 50
Fusarium TTACTAGGGATTCCTCGTTGANGAGCAATAATTGCAATGNTCTATCCCCA 50
Clone 10 TTACTAGGCATTCCTCGTTGAAGAGCANTAATTGCAATGNTCTATCCCCA 50
EP1 Probe-
EYESPOT GCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA-A 99 i — Septoria GCACGACGGAGTTTAACAAGATTACCCAGACCTTTCGGCCAAGGTGATGA 100 Fusarium GCACGANGGNGTTTNTCAAGNATACCCGGACCTTTCGGACAAGGA-AGT 98 Clone 10 GNACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGTGAAGA 100
EYESPOT ACTCGTCGGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAG 149
Septoria ACTCGTTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 149
Fusarium ACTCGCTGGCTCCGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 147
Clone 10 ACTCGTTGGCTCTGTCAGTGTAGCGCGCGTGCGGCCCAG-AACATCTAAG 149
EYESPOT GGCATCACAGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATA 199
Septoria GGCATCACAGACCTGTTATTGCCTCAAACTTCCATCGGCTTGAGCCGATA 199
Fusarium GGCATCACAGACCTGTTATTGCCTCAAACTTCCATCGGNTTGAGCCGATA 197
Clone 10 GGCATCACAGACCTGTTATTGNCTCAANCTTCCATCGGCTTGAGCCGATA 199
Nhel
EYESPOT GTCCCTCTAAGAAGCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCA 249
Septoria ■GTCCCTCTAAGAAGCCGGCGACCCGCCAAAGCGGGCCTGGCTATTTAGCA 249
Fusarium GTCCCTCTAAGAAGCCAGCGTACTGCCAAAGCAATACGGGCTATTTAGCA 247
Clone 10 GTCCCTCTAAGAAGCCGGTGACCAGCCAAAGCTAGCCTGGCTATTTAGCA 249
EYESPOT GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAANTCACTCCACCA 299
Septoria GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 299
Fusarium GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 297
Clone 10 GGTTAAGGTCTCGTTCGTTATCGCAATTAAGCAGACAAATCACTCCACCA 299
EYESPOT ACTAAG-AACGGTCATGC-ACCACCACCCACAAAATCAAGAAAGAGCTCT 347 Septoria ACTAAG-AACGGTCATGN- ACCACCACCCACAAAATCAAGAAAGAGCTCT 347
Fusarium ACTAAG-AACGGCCATGC- ACCACCACCCACAAAATCAAGAAAGAGCTCT 345
Clone 10 ACTANGTAACGGNCATGNNACCACCACCTGAAAAATCAAGAAAGAGNTCT 349
Example 1 1
IN PLANTA hybridization analysis
Amplifications and hybridizations were performed on DNA extracted from wheat, eyespot infected wheat, and Rhizoctonia cerealis. Amplification products were run on gels and southern blot was hybridized with the EP1 eyespot probe (see Fig. 7).
The hybridization conditions were 1 hour at 50°C (in a 5x SSC buffer, 0.5% SDS and 5x Denhardt buffer).
The EP1 probe hybridized to infected wheat, eyespot, but not with Rhizoctonia cerealis nor with Septoria or Fusarium, as noted above.
The results show that EP1 is a probe specific for eyespot which does not hybridize with Fusarium, Septoria, Rhizoctonia, nor with wheat itself. In terms of specificity, and considering the small number of various species analyzed, this probe can be considered as 100% specific. In terms of range of selectivity, and considering the
28 different strains tested, the probe can be considered as 96.5 % selective.
Description of the Figures
Fig. 1 shows the gel after the amplification of ribosomal RNA genes of eyespot, Fusarium and Septoria.
The samples were loaded onto a 0.8 % agarose gel and electrophoretically separated. The DNA was visualized by ethidium bromide (0.5 mg/ml in agarose gel) under U.V. The marker of the DNA is I (lambda) DNA digested by EcoRI and Hindlll.
Fig. 2 shows the EcoRI I Hindlll cleavage sites on the 1.9 kb long DNA-fragment with the position of the two primers (P1 and P2).
Fig. 3 shows the Southern blot of miniprep DNAs (from the positive clones) hybridized with probe P2 and probe P1 . Probe P2 is specific for the 0.3 kb EcoRI-Hindlll fragment of Eyespot and the 2 kb EcoRI-Hindlll fragment of Fusarium and Septoria. Probe P1 is specific for the 1 .6 kb EcoRI fragment of eyespot.
Fig. 4 shows the respective positions of primers P2 and P6, EcoRI and Nhel restriction sites and EP1 probe.
Fig. 5 shows the gel after the amplification of various strains of eyespot, Fusarium and Septoria DNA using both Primers 2 and 6.
The samples were electrophoretically separated on a 1 % agarose gel, and visualized by ethidium bromide (0.5μg/ml in the agarose gel). The marker is λ (lambda) DNA digested by EcoRI-Hindlll.
The numbers indicate the various strains of eyespot as follows:
1 : WF 447 10 : WUK 182 19 : : RF 988
2 : WF 927 11 : : WUK 170 20: : RG 349
3 : WF 611 12 : : WG 414 21 : : RG 301
4 : WUK 10 13 : : WG 142 22 : : RD 1545
5 : WF1000 14 : : WD 1816 23 : : RG 397
6 : : WUK 38 15 : : WD 1548 24 : : RD 1646
7 : WUK 20 16 : RUK 391 25 : : RD 1625
8 : : WG 112 17 : : RUK 717 26 : :RD 1711
9 : : WG 71 18 : : RF986 27 : : RD 2000
T = PCR negative control
Fig.6 shows the results of the hybridization of the amplified products with the eyespot probe, EP1.
Fig.7 shows the analysis of the hybridization in plants.
Claims
1. An oligonucleotide sequence as a probe specific for eyespot which does not hybridize with other phytopathogenic fungi or with cereals.
2. A probe as claimed in claim 1 , wherein the oligonucleotide sequence contains 16 to 20 nucleotides.
3. A probe as claimed in claim 1 or 2, wherein the oligonucleotide sequence is as follows:
5' TCAAGGAAGAAACTCGTC 3'
4. An oligonucleotide sequence as amplification primer for hybridization of the DNA-strains of Fusarium, Septoria and eyespot.
5. A primer as claimed in claim 4, wherein the oligonucleotide sequence contains 25 to 30 nucleotides.
6. A primer as claimed in claim 4 or 5, wherein the oligonucleotide sequence is as follows:
5' GCCGTCGACCAATTGTCAGAGGTGAAA 3'
7. An eyespot DNA having the following sequence:
GCCCGGGATCCAAGCTTGATCCTTCTGCAGGTTCACCTACGGAAACCTTGTTACG 55
ACTTTTACTTCCTCTAAATGACCAAGTTTGTACGATTTTCCAGCCCTGAGTGGGT 1 10 GTTGCCACCCTCTCTGGGCCGGTCCAAAGGCCTCACTTAGCCATTCAATCGGTAG 165 TAGCGACGGGCGGTGTGTACAAAGGGCAGGG-ACGTAATCAGCGCAAGCTGATGA 219
fEcoRI — j
CTTGCGCTTACTAGGAATTCCTCGTTGAAGAGCAATAATTGCAATGCTCTATCCC 274
r P1 Probe —
CAGCACGACAGAGTTTAACAAGATTACCCAGACCTTTCGGTCAAGGAAGA- AACT 328 CGTCGGCTCTGTCAGTGTAGCGCGCGTGCGGNCCAGNAACATCTAAGGGCATCAC 383 AGACCTGTTATTGTCTCAAACTTCCATCGGCTTGAGCCGATAGTCCCTCTAAGAA 438
rNhel — r GCCGGNGANCAGCCAAAGCTAGCCTGGCTATTTAGCAGGTTAAGGTCTCGTTCGT 493
TATCGCAATTAAGCAGACAANTCACTCCACCAACTAAGAACGGTCATGCACCACC 548
ACCCACAAAATCAAGAAAGAGCTCTCAATCTGTCAATCCTTATTGTGTCTGGACC 603
TGGTGAGTTTCCCCGTGTTGAGTCAAATTAAGCCGCAGGCTCCACTCCTGGCGGT 658
GNCCTTCCGTCAATTTCTTTAAGTTTCAGNCTTGGG ACCATACTCCCCCCAGTAA 713 CCCAAAGACTTTGNTTTCTCGTAAGGTGCCGAGCGAGTCAAAAAAATAACATTGC 768
CCGATCCCTAGTCGGCATAGTTTATGGTTAAGACTACGACGGTATCTGATCGTCT 823
TCGATCCCCTAACTTTCGGTCACTGTATTAATGAAAACATCCTTGGCAAATGCTT 878
TCGCAGNAGTTAGTCTTCAATAAATCCAAGTAATTTCACCTCTGACAATTGTAAT 933
8. Use of the oligonucleotide sequence according to any one of claims 1 to 3, for the identification of eyespot.
9. Use of the oligonucleotide sequence according to any one of claims 4 to 6, as primers for hybridization of Fusarium-, Septoria- and eyespot-DNA.
10. A diagnostic kit for the in vivo detection of eyespot infection in plants which comprises a probe according to any one of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP94909031A EP0686201A1 (en) | 1993-02-27 | 1994-02-19 | Eyespot dna-sequence |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB939304026A GB9304026D0 (en) | 1993-02-27 | 1993-02-27 | Eyespot method |
| GB9304026.9 | 1993-02-27 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1994019489A2 true WO1994019489A2 (en) | 1994-09-01 |
| WO1994019489A3 WO1994019489A3 (en) | 1994-10-27 |
Family
ID=10731175
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1994/000516 Ceased WO1994019489A2 (en) | 1993-02-27 | 1994-02-19 | Eyespot dna-sequence |
Country Status (3)
| Country | Link |
|---|---|
| EP (1) | EP0686201A1 (en) |
| GB (1) | GB9304026D0 (en) |
| WO (1) | WO1994019489A2 (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996029432A1 (en) * | 1995-03-22 | 1996-09-26 | Trustees Of Boston University | Methods for the detection of paracoccidioides |
| DE19615934C1 (en) * | 1996-04-22 | 1997-10-02 | Martin Ludwig Dr Niessen | Detection of Fusarium graminearum |
| DE19628959A1 (en) * | 1996-07-18 | 1998-01-22 | Martin Ludwig Dr Niessen | Detecting fungi which may produce trichothecene mycotoxins |
| US6500382B2 (en) | 2000-03-24 | 2002-12-31 | Sms Demag Ag | Method and apparatus for slag-free teeming of metal melt from a metallurgical melt vessel |
| US6821770B1 (en) * | 1999-05-03 | 2004-11-23 | Gen-Probe Incorporated | Polynucleotide matrix-based method of identifying microorganisms |
-
1993
- 1993-02-27 GB GB939304026A patent/GB9304026D0/en active Pending
-
1994
- 1994-02-19 WO PCT/EP1994/000516 patent/WO1994019489A2/en not_active Ceased
- 1994-02-19 EP EP94909031A patent/EP0686201A1/en not_active Withdrawn
Non-Patent Citations (3)
| Title |
|---|
| GENE vol. 71 , 1988 , HOLLAND pages 491 - 499 MEDLIN, L. ET AL. 'the characterization of enzymically amplified eukaroyte 16S-like rRNA-coding regions' cited in the application * |
| JOURNAL OF GENERAL MICROBIOLOGY vol. 138, no. 11 , November 1992 , GB pages 2305 - 2309 THOMAS, D ET AL. 'Indentification of rye- and wheat-types of Pseudocersporella herpotrichoides with DNA probes' * |
| PLANT PATHOLOGY vol. 41 , 1992 , GB pages 591 - 599 PRIESTLEY, R. ET AL 'Comparison of isoenzyme and DNA markers for differentiating W-, R- and C-pathotypes of Pseudocercosporella herpotrrichiodes.' * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996029432A1 (en) * | 1995-03-22 | 1996-09-26 | Trustees Of Boston University | Methods for the detection of paracoccidioides |
| DE19615934C1 (en) * | 1996-04-22 | 1997-10-02 | Martin Ludwig Dr Niessen | Detection of Fusarium graminearum |
| DE19628959A1 (en) * | 1996-07-18 | 1998-01-22 | Martin Ludwig Dr Niessen | Detecting fungi which may produce trichothecene mycotoxins |
| DE19628959B4 (en) * | 1996-07-18 | 2004-07-08 | Biotecon Diagnostics Gmbh | Use of the nucleotide sequence and the protein structure of enzymes and regulatory proteins of trichothecene biosynthesis for the qualitative and quantitative analytical detection of fungi with the potential to form trichothecene mycotoxins |
| US6821770B1 (en) * | 1999-05-03 | 2004-11-23 | Gen-Probe Incorporated | Polynucleotide matrix-based method of identifying microorganisms |
| US7449328B2 (en) | 1999-05-03 | 2008-11-11 | Gen-Probe Incorporated | Probe matrix-based device for identifying microorganisms |
| US6500382B2 (en) | 2000-03-24 | 2002-12-31 | Sms Demag Ag | Method and apparatus for slag-free teeming of metal melt from a metallurgical melt vessel |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9304026D0 (en) | 1993-04-14 |
| WO1994019489A3 (en) | 1994-10-27 |
| EP0686201A1 (en) | 1995-12-13 |
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