WO2006005770A2 - Method for the detection of fusarium graminearum - Google Patents

Method for the detection of fusarium graminearum Download PDF

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Publication number
WO2006005770A2
WO2006005770A2 PCT/EP2005/053394 EP2005053394W WO2006005770A2 WO 2006005770 A2 WO2006005770 A2 WO 2006005770A2 EP 2005053394 W EP2005053394 W EP 2005053394W WO 2006005770 A2 WO2006005770 A2 WO 2006005770A2
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Prior art keywords
nucleic acid
seq
acid sequence
reverse primer
tubulin
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PCT/EP2005/053394
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French (fr)
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WO2006005770A3 (en
Inventor
Robert Mach
Andreas Farnleitner
Georg Reischer
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Technische Universität Wien
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Priority to DE602005027841T priority Critical patent/DE602005027841D1/en
Priority to EP05778976A priority patent/EP1766083B1/en
Priority to CA002569190A priority patent/CA2569190A1/en
Priority to AT05778976T priority patent/ATE508204T1/en
Priority to US11/570,226 priority patent/US7919243B2/en
Publication of WO2006005770A2 publication Critical patent/WO2006005770A2/en
Publication of WO2006005770A3 publication Critical patent/WO2006005770A3/en

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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
    • 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 methods and kits for the detection of Fusarium graminearum (Gibberella zeae) .
  • Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schw.) Petch) is an important pathogen of cereal crops, causing root rot and seedling diseases (Cook, 1968; Manka et al., 1985), head blight of wheat and barley (Stoyan et al., 2003), and stalk and ear rot of maize (Cook, 1981; Kommedahl and Windels, 1981) . Fusarium head blight and ear rot reduce grain yield and the har ⁇ vested grain is often contaminated with mycotoxins such as tri- chothecenes and zeralenone (Lee et al. , 2002) . Trichothecene contamination is associated with feed refusal, vomiting, diarrhoea, dermatitis and haemorrhages (Marasas et al. , 1984) .
  • WO 99/07884 Al as well as WO 03/027635 A2 disclose methods for the detection of different fungal pathogenes, including Fusarium species, by using a PCR-based technique.
  • the primers employed in these methods are derived from Internal Transcribed Spacer (ITS) DNA sequences of the nuclear ribosomal RNA gene (rDNA) or from the mitochondrial Small Subunit Ribosomal DNA se ⁇ quences.
  • ITS Internal Transcribed Spacer
  • genes suitable for the characterisation of fungal pathogens comprise the ⁇ -tubulin gene.
  • This gene is highly con ⁇ served as compared to functional genes and thus allows the de ⁇ velopment of primers and probes that can reach all members of a group of phylogenetically related organisms at any level (Atkins and Clark, 2004; McCartney et al. , 2003) .
  • the sequence of the ⁇ -tubulin genes of Fusarium sp. is variable enough to allow distinction between fungi on the species level, which was a requirement for the species-specific detection of Fusarium graminearum.
  • rRNA genes ri ⁇ bosomal RNA
  • ITS Internal Tran ⁇ scribed Spacer regions
  • US 5,707,802 discloses a PCR method for the detection of pa ⁇ thogenic fungus strains by employing primers specific for a hyper variable region of 28S rDNA or rRNA.
  • DE 196 15 934 relates to a method for detecting Fusarium graminearum in a sample wherein said detection occurs by the de ⁇ termination of the enzymatic activity of galactose oxidase, binding of antibodies to said oxidase or by using primers spe ⁇ cific for the nucleotide sequence of galactose oxidase in a PCR reaction.
  • Knoll et al. disclose a PCR based method for the detection of galactose oxidase of Fusarium graminearum in a sample.
  • Hue et al. J Clin Microbiol (1999) 37:2434-2438) describe the specific detection of Fusarium strains in blood or tissue samples by using an rDNA specific PCR.
  • the present invention provides a method for the detection of Fusarium graminearum (Gibberella zeae) comprising the steps:
  • the at least one reverse primer hybridises within the ⁇ -tubulin nucleic acid sequence of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence (SEQ ID NO 1), wherein R 1 comprises at least one nucleic acid of the ⁇ -tu ⁇ bulin nucleic acid sequence located upstream of the hybrid ⁇ isation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid sequence SEQ ID NO 1, X x is guanine, adenine or inosine, X 2 is cytosine, adenine or in- osine, and wherein the at least one forward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer,
  • Providing a sample containing a nucleic acid refers to methods known in the state of the art, which can be employed to isolate a sample containing a nucleic acid.
  • nucleic acids can be further isolated from a sample containing nucleic acids, for instance according to methods disclosed in Sambrook, J., and Russell, D. W. (2001) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • nucleic acid amplification techniques e.g. in situ PCR
  • nucleic acid and “ ⁇ -tubulin nucleic acid sequence” refer to DNA as well as to RNA and mRNA.
  • sample includes all type of samples, which may be infected by fungi, especially by F. graminearum (G. zeae) . Therefore a sample according to the present invention comprises e.g. cereals and food and food products derived from cereals, soil, garden soil products, animal feed, infected plant materi ⁇ al, compost, fungal spores from air, rain and hail.
  • F. graminearum G. zeae
  • Hybridising is the ability of primers consisting of nucle ⁇ ic acids to bind specifically to a nucleic acid sequence under stringent conditions and under conditions applied in the course of a polymerase chain reaction. Stringent conditions are se ⁇ quence-dependent and will be different in different circum ⁇ stances. Longer sequences hybridise specifically at higher temperatures. An extensive guide to the hybridisation of nucleic acids is found in Tijssen, Techniques in Biochemistry and Mo ⁇ lecular Biology—Hybridisation with Nucleic Probes, "Overview of principles of hybridisation and the strategy of nucleic acid as ⁇ says” (1993) .
  • stringent conditions are selected to be about 5 to 10 0 C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm is the temperature (under defined ionic strength, pH, and nucle ⁇ ic acid concentration) at which 50% of the probes complementary to the target hybridise to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium) .
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion con ⁇ centration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (e.g. 10 to 50 nucle ⁇ otides) and at least about 6O 0 C for long probes (e.g. greater than 50 nucleotides) .
  • the term "at least one" clarifies that according to the present invention one or more forward or reverse primers can be employed in a single or in a multiple series of polymerase chain reactions.
  • Some polymerase chain reaction techniques e.g. nes ⁇ ted PCR, TaqMan-PCR, employ more than one forward and/or more than one reverse primers.
  • ⁇ -tubulin nucleic acid sequences are publicly available from databases like the NCBI (http://www.ncbi.nlm.nih.gov/en-technische) or the EMBL database (http://www.ch.embnet.org/software/bBLAST.html) .
  • a primer comprising the sequence 5'-R 1 TTTTCGTX 2 GX 3 AGT-S' can be used as reverse primer in combination with an appropriate forward primer in a polymerase chain reaction in or ⁇ der to obtain a specific fragment. If such a fragment is ob ⁇ tained, F. graminearum (G. zeae) is present in the sample analyzed.
  • F. graminearum G. zeae
  • This specificity on the species level is achieved by utilising a single diagnostic base (the thymine residue at the 3'-end) that is unique for the ⁇ -tubulin gene of F. graminearum and is not found in other Fusarium species at this position in the gene. This is the only position in the ⁇ -tubulin gene's chain 1 where such an exclusive distinction is possible.
  • the re ⁇ verse primer selectively binds to this diagnostic base. Posi ⁇ tioning the primers at any different site would lead to loss of selectivity.
  • the at least one reverse primer and/or the at least one forward primer comprise (s) 14 to 50, preferably 16 to 45, more preferably 18 to 40, more prefer ⁇ ably 19 to 35, especially 20 to 30, nucleic acid residues.
  • the length of the at least one primer which can influence its bind ⁇ ing specificity to the nucleic acid template, can be varied de ⁇ pending on the nucleic acid amplification technique and reaction conditions.
  • the at least one reverse primer is selected from the group consisting of 5'-GCTTGTGTTTTTCGTGGCAGT-S' (SEQ ID NO 2), 5'-GCTTGTGTTTTTCGTAGCAGT-S' (SEQ ID NO 3),
  • the nucleic acid amplification technique is preferably a polymerase chain reaction technique.
  • the polymerase chain re ⁇ action technique is selected from the group consisting of real ⁇ time PCR, preferably TaqMan PCR, quantitative PCR, nested PCR, assymetric PCR, multiplex PCR, inverse PCR, rapid PCR and com ⁇ binations thereof.
  • every polymerase chain reaction technique known in the state of the art can be used to detect F. graminearum (G. Zeae) .
  • the at least one forward primer hybridises within the ⁇ -tubulin cluster of Fusarium graminearum (Gibberella zeae) . Also forward primers hybridising not within the ⁇ -tubulin cluster can be used for detecting F. graminearum (G. Zeae) in a sample, if these primers are located upstream of the ⁇ -tubulin gene where the at least one reverse primer is derived from.
  • the at least one forward primer is derived from F. graminearum (G. zeae) and hybridises in a distance of at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500 or at least 1000 nucleic acid residues to the at least one reverse primer.
  • Forward primers can easily be designed on the basis of the DNA template•
  • the nucleic acid frag ⁇ ment obtained by the nucleic acid amplification according to the present invention comprises at least 50, at least 80, at least 110, at least 150, at least 200, at least 300, at least 400, at least 500 or at least 1000 nucleic acids.
  • the at least one forward primer comprises the sequence S'-GGTCTCGACAGCAATGGTGTT- 3' (SEQ ID NO 6) or 5'-GGTCTTGACAGCAATGGTGTT-3' (SEQ ID NO 7) . Both primers hybridise within the ⁇ -tubulin cluster of F. graminearum (G. zeae) and are located upstream of the at least one reverse primer.
  • the polymerase chain reaction is performed with at least one oligonucleotide probe hybridising in between of the at least one forward primer and the at least one reverse primer within the ⁇ -tubulin cluster of Fusarium graminearum (Gibberella zeae) .
  • the at least one oligonucleotide probe is tagged with a dye, preferably a fluorescent dye, and optionally with a quencher.
  • a dye preferably a fluorescent dye
  • An additional oligonucleotide which hybridises between the at least one for ⁇ ward and the at least reverse primer within the ⁇ -tubulin cluster and is tagged with a dye and a quencher (e.g. FAM/TAMRA) , is added to the PCR reaction mixture in order to visualise the nucleic acid fragment already during the synthesis in a thermocycler (quantitative PCR) .
  • FAM/TAMRA quencher
  • the addition of another oligonucleotide enhances the specificity and yield of a PCR.
  • the at least one oligonucleotide probe comprises the sequence 5'-ACAACGGCACCTCTGAGCTCCAGC-3' (SEQ ID NO 8) or 5'-ACAACGGTACCTCTGAGCTCCAGC-3' (SEQ ID NO 9) .
  • the amplified nucleic acid fragment is additionally tagged for visualisation by a technique selected from the group consisting of DNA-tagging by random-priming, DNA-tagging by nick-transla ⁇ tion, DNA-tagging by polymerase chain reaction, oligonucleotide tailing, hybridisation, tagging by kinase activity, fill-in re ⁇ action applying Klenov fragment, photobiotinylation and combina ⁇ tions thereof. Due to a tagging of the PCR product this product can easily be visualised by an additional further step involving e.g. gel electrophoresis.
  • the amplified nucleic acid fragment is visualised by gel electrophoresis, Southern-blot, photometry, chromato ⁇ graphy, colorimetry, fluorography, chemoluminescence, autoradio ⁇ graphy, detection by specific antibody and combinations thereof.
  • the present invention relates to a kit for the detection of Fusarium graminearum (Gibberella zeae) comprising at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybrid ⁇ ises within the ⁇ -tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-R I TTTTCGTX 1 GX 2 AGT-S 1 (SEQ ID NO 1), wherein R 1 comprises at least one nucleic acid residue of the ⁇ -tubulin nucleic acid se- quence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid se ⁇ quence SEQ ID NO 1, Xi is guanine, adenine or inosine, X 2 is cytosine, adenine or inosine, and wherein the at least one for ⁇ ward primer hybridises upstream of the hybridisation site
  • the present invention relates to a use of at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises with ⁇ in the ⁇ -tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-R I TTTTCGTX 1 GX 2 AGT-S' (SEQ ID NO 1), wherein R 1 comprises at least one nucleic acid residue of the ⁇ -tubulin nucleic acid se ⁇ quence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid se ⁇ quence SEQ ID NO 1, Xi is guanine, adenine or inosine, X 2 is cytosine, adenine or inosine, and wherein the at least one for ⁇ ward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer, for the detection of Fusarium
  • the developed primers and probes may also serve as basis for other detection methods such as SYBR green real-time PCR (using only the diagnostic primer pair) , other probe dependent real ⁇ time PCR methods e.g. Molecular Beacon or Scorpion primer- probes.
  • SYBR green real-time PCR using only the diagnostic primer pair
  • probe dependent real ⁇ time PCR methods e.g. Molecular Beacon or Scorpion primer- probes.
  • the applied probe se ⁇ quence can be utilised for the design of a FISH (Fluorescent In- Situ Hybridisation) probe used to monitor the propagation of the plant pathogen in the respective plant tissue.
  • FISH Fluorescent In- Situ Hybridisation
  • the chosen diagnostic fragment bears the potential for designing primer/probe combinations for a species-specific de ⁇ tection of additional Fusarium species, especially other head blight agents.
  • Fig.l shows ⁇ -tubulin gene copy numbers per mg (cn/mg) of plant material (wet weight) detected with the real-time PCR as ⁇ say in samples taken from the inoculation experiment before in ⁇ fection (bi) and during all stages of infection 1, 2, 4, 6, 8 and 16 days post infection (dpi) .
  • Columns represent the median value of 27 seperate measurements for each day, error bars in ⁇ dicate the maximum and minimum values, respectively. Below, pic ⁇ tures of the respective wheat spikes are shown.
  • Fig.2 is a schematic drawing of the diagnostic fragment of the ⁇ -tubulin gene.
  • Various Fusarium ⁇ -tubulin gene sequences (taken from publicly available databases; database accession numbers are cited next to the organism name) were aligned (using the Vector NTI software package) to identify a species specific primer/probe combination highlighted in green boxes.
  • the species specific reverse primer FGtubr is positioned within the third intron of the ⁇ -tubulin gene, the species specific base is situ ⁇ ated at the 3 f end of the primer (indicated with an arrow) .
  • the positioning of the diagnostic fragment is given in the figure and is related to the Fusarium graminearum ⁇ -tubulin sequence.
  • EXAMPLE Species-specific identification and absolute quan ⁇ tification of F. graminearum in planta by a real-time PCR assay using a TaqMan hybridisation probe.
  • the method according to the present invention was tested on plant material from wheat artificially infected in open field experiment.
  • DNA of pure cultures from the strain collections of the In ⁇ stitute of Chemical Engineering and the IFA Tulln was extracted following the method previously described by Ari- san-Atac et al., 1995.
  • DNA from wheat spikes of defined weight was obtained by grinding of whole spikes in liquid nitrogen, taking an aliquot of 50 ⁇ g from the homogenate and subsequent DNA extraction as described by Arisan-Atac et al.
  • a bead-beating step using a Fast-Prep FP 120 (Thermo Savant, Holbrook, NY) at an intensity setting of 6 for 30 sec was introduced after addition of CTAB buffer, fol ⁇ lowed by a phenol-chloroform-isoamyl alcohol (25:24:1) extrac ⁇ tion (Griffiths et al. , 2000) .
  • the purified DNA was resuspended in 50 ⁇ l of sterile distilled water and stored at -8O 0 C. DNA ex ⁇ traction from wheat spikes was done from 3 different aliquots of each spike to compensate for variations in extraction effi ⁇ ciency.
  • avenacea [6] were extracted from the NCBI database and aligned using the Vector NTI software package (InforMax Inc., Frederick, MD) . Primers were designed from the derived consensus sequence with the Primer Express ® software (Ap ⁇ plied Biosysterns, Foster City, CA) . The primers FGtubf (5'-GGTCTCGACAGCAATGGTGTT-S') and FGtubr
  • a 570 bp fragment of the beta-tubulin gene of F. graminearum isolate IFA 66 encom ⁇ passing the target region of the real-time assay was cloned into a pGEM-T Vector (Promega, Madison, WI) .
  • the concentration of the plasmid standard solution was measured photometrically and the standard was diluted in lOfold steps in a 10 ng/ ⁇ l poly [d(I-C)] solution as unspecific DNA background (Roche Diagnostics, Mannheim, Germany) .
  • PCR was monitored on an iCycler iQ Real-Time Detection Sys ⁇ tem (Biorad, Hercules, CA) .
  • Reaction mixtures (25 ⁇ l total volume) contained 2.5 ⁇ l of template DNA dilution, 7.5 pmol of FGtubf, 7.5 pmol of FGtubr, 6.25 pmol of FGtubTM, 12.5 ⁇ l of iQ Supermix (Biorad) and 10 ⁇ l of sterile bi-distilled water.
  • the PCR programme was the following: 95°C for 3 min 30 sec (denatur- ation, activation of polymerase, measuring of well factors), 40 cycles of 95°C for 15 s, 67°C for 15 s and 72 0 C for 20 s.
  • IFA 191 IFA maize ker ⁇ AY629347 + + nel a ICE, Fusarium strain collection Institute of Chemical Engineer ⁇ ing, Vienna, Austria; IFA strain collection of IFA, Tulln, Aus ⁇ tria.
  • IFA 191 isolate F. graminearum isolated from a Triticum durum kernel in 1990 in Austria
  • a modified Czapek-Dox medium (con ⁇ taining/1 of bi-distilled water in g: glucose, 20; KH 2 PO 4 , 0.5; NaNO 3 , 2; MgSO 4 .7H 2 O, 0.5; yeast extract, 1 and 1 ml of a 1% FeSO 4 solution (w/w) ) supplemented with 0.1 g streptomycin sulphate/1 and 0.01 g neomycin/1 after autoclaving was used for inoculum production. After 1 week the mycelium suspension was ready and 300 ml of the suspension was diluted in 1 1 of bi-distilled wa ⁇ ter for inoculation.
  • the wheat line E2-23-T was grown in 2 m 2 plots at the IFA experimental field.
  • This wheat line is a doubled haploid line originating from a cross between CM82036 and Remus and was pro ⁇ quizled with the maize-wheat pollination system. It is a very sus ⁇ ceptible line and does not have a quantitative trait locus (QTL) for Fusarium head blight resistance on chromosome 5A and 3B (Buerstmayr et al., 2002; Buerstmayr et al., 2003) .
  • QTL quantitative trait locus
  • the wheat line was inoculated at 50% anthesis by spraying 100 ml/m 2 of the inoculum suspension described above. To promote infection an automated mist irrigation system was used to apply moisture. Water was applied in 2 pulses of 10 s each at 15 min intervals during 17 hours after inoculation. Before inoculation 10 flowering spikes were cut and removed from the plot. On day 1, 2, 4, 6, 8 and 16 after inoculation the sampling procedure was repeated. All spikes were immediately stored at -8O 0 C after sampling for further investigations.
  • the primers, probe and PCR protocol were evaluated by ampli ⁇ fying DNA from pure culture isolates of F. gram ⁇ nearum as well as the developed plasmid standard. Specificity was assessed by amplification of DNAs from various isolates which are either closely related to F. graminearum and/or also causing head blight in wheat.
  • the ⁇ -tubulin gene of all non F. gram ⁇ nearum isolates failed to be amplified in the reaction while DNA from all isolates yielded products in the general Fusarium PCR assay (Table 1) . All PCR products were of the expected size when ex ⁇ amined by agarose gel electrophoresis.
  • the efficiency of the amplification was in ⁇ vestigated using plasmid standard as well as known amounts of F. graminearum sample isolate DNA as template in the presence of unspecific DNA, such as the DNA from isolates closely related to F. graminearum (Table 1) and DNA extracted from uninfected wheat spikes. No change in threshold cycle values could be observed under these conditions as compared to amplification without a DNA background. In addition the overall efficiency of the PCR amplification was very high regardless of template origin or un ⁇ specific DNA background (95-98.8%) .
  • the developed method was applied on the total DNA extracted from spikes of infected wheat plants in all states of infection.
  • the ⁇ -tubulin gene could not be detected in samples from unin ⁇ fected wheat. However, detection was possible in all samples taken after inoculation with F. graminearum isolate IFA 191. There was a distinct gradual increase in gene copies measured, corresponding to the fungal growth during the infection of the wheat spike (Fig. 1) . Detection was possible from the first day post inoculation, while first symptoms of the infection (water soaked spots) appeared 7 to 10 days after inoculation (see Fig. 1) . Data of all aliquots, dilution steps and replicates are in ⁇ cluded in Fig.l.
  • the method according to the present invention allows a fast, species-specific identification and quantitation of plant-infec ⁇ tions by F. graminearum at very early stages where classical mi ⁇ crobiological and toxin analysis methods fail to detect the pathogen (McCartney et al., 2003) . It can be applied on DNA ex ⁇ tracted directly from presumptively infected plant material and is not affected by an unspecific background of either plant or fungal DNA, even from other pathogens causing head blight. It allows a reliable estimation of the fungal genomes and can de ⁇ tect as few as 5 gene copies. The assay is cheaper and less time consuming than microbiological identification methods.
  • the presented method will be an invaluable tool for the study of F. graminearum infection processes.
  • the sensitivity of the method would even allow the quantitation of fungal growth in different plant tissues during the progress of infection (Stoyan et al., 2003) . Due to its simplicity it could also be used in routine analysis and the monitoring of F. graminearum epidemics or the monitoring of pest control meas ⁇ ures.
  • Buerstmayr H., Lemmens, M., Hartl, L., Doldi, L., Steiner, B., Stierschneider, M., Ruckenbauer, P., 2002. Theor. Appl. Genet. 104, 84-91.

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Abstract

A method for the detection of Fusarium graminearum (Gibberella zeae) comprising the steps: providing a sample containing a nucleic acid, contacting said sample with at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises within the ß-tubulin nucleic acid sequence of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-R1TTTTCGTX1GX2AGT-3' (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the ß-tubulin nucleic acid sequence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid sequence SEQ ID NO 1, X1 is guanine, adenine or inosine, X2 is cytosine, adenine or inosine, and wherein the at least one forward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer, subjecting the sample contacted with the at least one forward primer and the at least one reverse primer to a nucleic acid amplification technique, and optionally determing the presence of Fusarium graminearum (Gibberella zeae) in a sample by detecting a nucleic acid amplification product.

Description

Method for the Detection of Fusarium graminearum
The present invention relates to methods and kits for the detection of Fusarium graminearum (Gibberella zeae) .
Fusarium graminearum Schwabe (teleomorph: Gibberella zeae (Schw.) Petch) is an important pathogen of cereal crops, causing root rot and seedling diseases (Cook, 1968; Manka et al., 1985), head blight of wheat and barley (Stoyan et al., 2003), and stalk and ear rot of maize (Cook, 1981; Kommedahl and Windels, 1981) . Fusarium head blight and ear rot reduce grain yield and the har¬ vested grain is often contaminated with mycotoxins such as tri- chothecenes and zeralenone (Lee et al. , 2002) . Trichothecene contamination is associated with feed refusal, vomiting, diarrhoea, dermatitis and haemorrhages (Marasas et al. , 1984) .
Up to date the identification of the pathogen is relying on conventional methods like the interpretation of visual symptoms or the isolation and culturing of the fungus. The drawback of those methods is that detection of the pathogen is only possible in late stages of the infection when it is already too late for any countermeasures and the spread of the disease cannot be con¬ trolled anymore. In contrast, molecular diagnostics of plant pathogenic fungi can be highly specific, very sensitive and rel¬ atively fast (McCartney et al. , 2003) .
In the state of the art several methods are disclosed for the detection of F. Graminearum. For instance Edwards et al. (2001) describe a PCR-based method for the identification and the quantification of several Fusarium species, including F. Graminearum, within harvested grain. For the identification as well as for the quantification primers directed to the tricho- diene synthese gene (Tri5) were designed and used in a diagno¬ stic and in a quantitative PCR. The method disclosed in this article allows to distinguish Tri5 harbouring Fusarium species from species lacking of Tri5. Therefore an accurate distinction of Fusarium species is not possible, because the Tri5 gene is widely spread among Fusarium species and is not a characteristic feature of a single species.
WO 99/07884 Al as well as WO 03/027635 A2 disclose methods for the detection of different fungal pathogenes, including Fusarium species, by using a PCR-based technique. The primers employed in these methods are derived from Internal Transcribed Spacer (ITS) DNA sequences of the nuclear ribosomal RNA gene (rDNA) or from the mitochondrial Small Subunit Ribosomal DNA se¬ quences.
Other genes suitable for the characterisation of fungal pathogens comprise the β-tubulin gene. This gene is highly con¬ served as compared to functional genes and thus allows the de¬ velopment of primers and probes that can reach all members of a group of phylogenetically related organisms at any level (Atkins and Clark, 2004; McCartney et al. , 2003) . On the other hand the sequence of the β-tubulin genes of Fusarium sp. is variable enough to allow distinction between fungi on the species level, which was a requirement for the species-specific detection of Fusarium graminearum. As mentioned above another gene target commonly used in this type of assay, are the genes of the ri¬ bosomal RNA (rRNA genes) and their intervening Internal Tran¬ scribed Spacer regions (ITS) , which is too highly conserved in Fusarium sp. and does not allow a differentiation on this low level of phylogenetic affiliation (O'Donnell and Cigelnik, 1997) . In addition, both mentioned "phylogenetic" genes are well investigated and a large amount of sequence information from fungal isolates and environmental samples is available. That fa¬ cilitates the development of PCR primers and hybridisation probes and allows a better estimation of the selectivity of the developed tools. The use of the β-tubulin gene for phylogenetic studies and diagnostic applications is a well established prac¬ tice (Fraaije et al., 1998; O'Donnell et al. , 1998; O'Donnell et al., 2000; Yli-Mattila et al., 2004) .
US 5,707,802 discloses a PCR method for the detection of pa¬ thogenic fungus strains by employing primers specific for a hyper variable region of 28S rDNA or rRNA.
In the international patent application WO 2004/029216 a me¬ thod for detecting the presence of invasive pathogenic molds in a biological sample by detecting a 5.8S ribosomal RNA fragment of the respective mold via PCR is -disclosed.
DE 196 15 934 relates to a method for detecting Fusarium graminearum in a sample wherein said detection occurs by the de¬ termination of the enzymatic activity of galactose oxidase, binding of antibodies to said oxidase or by using primers spe¬ cific for the nucleotide sequence of galactose oxidase in a PCR reaction. Similarly, Knoll et al. (Lett Appl Microbiol (2002) 34: 144- 148) disclose a PCR based method for the detection of galactose oxidase of Fusarium graminearum in a sample.
Hue et al. (J Clin Microbiol (1999) 37:2434-2438) describe the specific detection of Fusarium strains in blood or tissue samples by using an rDNA specific PCR.
Jaeger et al. (J Clin Microbiol (2000) 38: 2902-2908) iden¬ tified members of Candida, Aspergillus and Fusarium species in a sample by using a nested PCR specific for a 18S rRNA fragment.
In providing suitable methods for specific detection of mi¬ croorganisms, techniques developed for strain typing are usually not exact enough for unambiguous identification of a single strain. This unambiguous identification requires identification of strain properties which are unique and which are identifiable by reproduceable and robust methods, especially when conducted on diverse sample material from natural sources. It is therefore an object of the present invention to provide suitable means for the specific detection of Fusarium graminearum (Gibberella zeae) in a sample allowing a clear differentiation from other Fusarium spieces.
Therefore the present invention provides a method for the detection of Fusarium graminearum (Gibberella zeae) comprising the steps:
- providing a sample containing a nucleic acid,
- contacting said sample with at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises within the β-tubulin nucleic acid sequence of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence
Figure imgf000004_0001
(SEQ ID NO 1), wherein R1 comprises at least one nucleic acid of the β-tu¬ bulin nucleic acid sequence located upstream of the hybrid¬ isation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid sequence SEQ ID NO 1, Xx is guanine, adenine or inosine, X2 is cytosine, adenine or in- osine, and wherein the at least one forward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer,
- subjecting the sample contacted with the at least one for¬ ward primer and the at least one reverse primer to a nucleic acid amplification technique, and - optionally determing the presence of Fusarium graminearum (Gibberella zeae) in a sample by detecting a nucleic acid amplification product.
"Providing a sample containing a nucleic acid" refers to methods known in the state of the art, which can be employed to isolate a sample containing a nucleic acid. However, nucleic acids can be further isolated from a sample containing nucleic acids, for instance according to methods disclosed in Sambrook, J., and Russell, D. W. (2001) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For certain nucleic acid amplification techniques (e.g. in situ PCR) the isolation of a nucleic acid from a sample is not necessary.
According to the present invention the terms "nucleic acid" and "β-tubulin nucleic acid sequence" refer to DNA as well as to RNA and mRNA.
The term "sample" includes all type of samples, which may be infected by fungi, especially by F. graminearum (G. zeae) . Therefore a sample according to the present invention comprises e.g. cereals and food and food products derived from cereals, soil, garden soil products, animal feed, infected plant materi¬ al, compost, fungal spores from air, rain and hail.
"Hybridising" is the ability of primers consisting of nucle¬ ic acids to bind specifically to a nucleic acid sequence under stringent conditions and under conditions applied in the course of a polymerase chain reaction. Stringent conditions are se¬ quence-dependent and will be different in different circum¬ stances. Longer sequences hybridise specifically at higher temperatures. An extensive guide to the hybridisation of nucleic acids is found in Tijssen, Techniques in Biochemistry and Mo¬ lecular Biology—Hybridisation with Nucleic Probes, "Overview of principles of hybridisation and the strategy of nucleic acid as¬ says" (1993) . Generally, stringent conditions are selected to be about 5 to 100C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucle¬ ic acid concentration) at which 50% of the probes complementary to the target hybridise to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium) . Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion con¬ centration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O0C for short probes (e.g. 10 to 50 nucle¬ otides) and at least about 6O0C for long probes (e.g. greater than 50 nucleotides) .
The term "at least one" clarifies that according to the present invention one or more forward or reverse primers can be employed in a single or in a multiple series of polymerase chain reactions. Some polymerase chain reaction techniques, e.g. nes¬ ted PCR, TaqMan-PCR, employ more than one forward and/or more than one reverse primers.
The β-tubulin nucleic acid sequences are publicly available from databases like the NCBI (http://www.ncbi.nlm.nih.gov/en- trez) or the EMBL database (http://www.ch.embnet.org/software/bBLAST.html) .
A primer comprising the sequence 5'-R1TTTTCGTX2GX3AGT-S' (SEQ ID NO 1) can be used as reverse primer in combination with an appropriate forward primer in a polymerase chain reaction in or¬ der to obtain a specific fragment. If such a fragment is ob¬ tained, F. graminearum (G. zeae) is present in the sample analyzed. This specificity on the species level is achieved by utilising a single diagnostic base (the thymine residue at the 3'-end) that is unique for the β-tubulin gene of F. graminearum and is not found in other Fusarium species at this position in the gene. This is the only position in the β-tubulin gene's chain 1 where such an exclusive distinction is possible. The re¬ verse primer selectively binds to this diagnostic base. Posi¬ tioning the primers at any different site would lead to loss of selectivity.
According to a preferred embodiment the at least one reverse primer and/or the at least one forward primer comprise (s) 14 to 50, preferably 16 to 45, more preferably 18 to 40, more prefer¬ ably 19 to 35, especially 20 to 30, nucleic acid residues. The length of the at least one primer, which can influence its bind¬ ing specificity to the nucleic acid template, can be varied de¬ pending on the nucleic acid amplification technique and reaction conditions.
According to another preferred embodiment the at least one reverse primer is selected from the group consisting of 5'-GCTTGTGTTTTTCGTGGCAGT-S' (SEQ ID NO 2), 5'-GCTTGTGTTTTTCGTAGCAGT-S' (SEQ ID NO 3),
5'-GCTTGTATTTTTCGTGGCAGT-S' (SEQ ID NO 4) and
5'-GCTTGTGTTTTTCGTGGAAGT-S' (SEQ ID NO 5). These reverse primers can alternatively be used, without loosing the specificity in detecting F. graminearum (G. Zeae) .
The nucleic acid amplification technique is preferably a polymerase chain reaction technique.
According to a preferred embodiment the polymerase chain re¬ action technique is selected from the group consisting of real¬ time PCR, preferably TaqMan PCR, quantitative PCR, nested PCR, assymetric PCR, multiplex PCR, inverse PCR, rapid PCR and com¬ binations thereof. According to the present invention every polymerase chain reaction technique known in the state of the art can be used to detect F. graminearum (G. Zeae) .
According to a preferred embodiment the at least one forward primer hybridises within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) . Also forward primers hybridising not within the β-tubulin cluster can be used for detecting F. graminearum (G. Zeae) in a sample, if these primers are located upstream of the β-tubulin gene where the at least one reverse primer is derived from.
Preferably the at least one forward primer is derived from F. graminearum (G. zeae) and hybridises in a distance of at least 25, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500 or at least 1000 nucleic acid residues to the at least one reverse primer. Forward primers can easily be designed on the basis of the DNA template•
According to a preferred embodiment the nucleic acid frag¬ ment obtained by the nucleic acid amplification according to the present invention comprises at least 50, at least 80, at least 110, at least 150, at least 200, at least 300, at least 400, at least 500 or at least 1000 nucleic acids.
According to another preferred embodiment the at least one forward primer comprises the sequence S'-GGTCTCGACAGCAATGGTGTT- 3' (SEQ ID NO 6) or 5'-GGTCTTGACAGCAATGGTGTT-3' (SEQ ID NO 7) . Both primers hybridise within the β-tubulin cluster of F. graminearum (G. zeae) and are located upstream of the at least one reverse primer.
Preferably the polymerase chain reaction is performed with at least one oligonucleotide probe hybridising in between of the at least one forward primer and the at least one reverse primer within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) .
According to a another preferred embodiment the at least one oligonucleotide probe is tagged with a dye, preferably a fluorescent dye, and optionally with a quencher. An additional oligonucleotide, which hybridises between the at least one for¬ ward and the at least reverse primer within the β-tubulin cluster and is tagged with a dye and a quencher (e.g. FAM/TAMRA) , is added to the PCR reaction mixture in order to visualise the nucleic acid fragment already during the synthesis in a thermocycler (quantitative PCR) . Furthermore the addition of another oligonucleotide enhances the specificity and yield of a PCR.
Preferably the at least one oligonucleotide probe comprises the sequence 5'-ACAACGGCACCTCTGAGCTCCAGC-3' (SEQ ID NO 8) or 5'-ACAACGGTACCTCTGAGCTCCAGC-3' (SEQ ID NO 9) .
According to a preferred embodiment of the present invention the amplified nucleic acid fragment is additionally tagged for visualisation by a technique selected from the group consisting of DNA-tagging by random-priming, DNA-tagging by nick-transla¬ tion, DNA-tagging by polymerase chain reaction, oligonucleotide tailing, hybridisation, tagging by kinase activity, fill-in re¬ action applying Klenov fragment, photobiotinylation and combina¬ tions thereof. Due to a tagging of the PCR product this product can easily be visualised by an additional further step involving e.g. gel electrophoresis.
Preferably the amplified nucleic acid fragment is visualised by gel electrophoresis, Southern-blot, photometry, chromato¬ graphy, colorimetry, fluorography, chemoluminescence, autoradio¬ graphy, detection by specific antibody and combinations thereof.
According to another aspect, the present invention relates to a kit for the detection of Fusarium graminearum (Gibberella zeae) comprising at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybrid¬ ises within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-RITTTTCGTX1GX2AGT-S1 (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the β-tubulin nucleic acid se- quence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid se¬ quence SEQ ID NO 1, Xi is guanine, adenine or inosine, X2 is cytosine, adenine or inosine, and wherein the at least one for¬ ward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer.
According to another aspect, the present invention relates to a use of at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises with¬ in the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-RITTTTCGTX1GX2AGT-S' (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the β-tubulin nucleic acid se¬ quence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid se¬ quence SEQ ID NO 1, Xi is guanine, adenine or inosine, X2 is cytosine, adenine or inosine, and wherein the at least one for¬ ward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer, for the detection of Fusarium graminearum (Gibberella zeae) .
The developed primers and probes may also serve as basis for other detection methods such as SYBR green real-time PCR (using only the diagnostic primer pair) , other probe dependent real¬ time PCR methods e.g. Molecular Beacon or Scorpion primer- probes. In addition it shall be noted that the applied probe se¬ quence can be utilised for the design of a FISH (Fluorescent In- Situ Hybridisation) probe used to monitor the propagation of the plant pathogen in the respective plant tissue.
The chosen diagnostic fragment bears the potential for designing primer/probe combinations for a species-specific de¬ tection of additional Fusarium species, especially other head blight agents.
The present invention is further illustrated by the follow¬ ing example and figures, yet without being restricted thereto.
Fig.l shows β-tubulin gene copy numbers per mg (cn/mg) of plant material (wet weight) detected with the real-time PCR as¬ say in samples taken from the inoculation experiment before in¬ fection (bi) and during all stages of infection 1, 2, 4, 6, 8 and 16 days post infection (dpi) . Columns represent the median value of 27 seperate measurements for each day, error bars in¬ dicate the maximum and minimum values, respectively. Below, pic¬ tures of the respective wheat spikes are shown.
Fig.2 is a schematic drawing of the diagnostic fragment of the β-tubulin gene. Various Fusarium β-tubulin gene sequences (taken from publicly available databases; database accession numbers are cited next to the organism name) were aligned (using the Vector NTI software package) to identify a species specific primer/probe combination highlighted in green boxes. The species specific reverse primer FGtubr is positioned within the third intron of the β-tubulin gene, the species specific base is situ¬ ated at the 3f end of the primer (indicated with an arrow) . The positioning of the diagnostic fragment is given in the figure and is related to the Fusarium graminearum β-tubulin sequence.
EXAMPLE: Species-specific identification and absolute quan¬ tification of F. graminearum in planta by a real-time PCR assay using a TaqMan hybridisation probe.
The method according to the present invention was tested on plant material from wheat artificially infected in open field experiment.
MATERIAL AND METHODS:
DNA of pure cultures from the strain collections of the In¬ stitute of Chemical Engineering and the IFA Tulln (see Table 1) was extracted following the method previously described by Ari- san-Atac et al., 1995. DNA from wheat spikes of defined weight was obtained by grinding of whole spikes in liquid nitrogen, taking an aliquot of 50 μg from the homogenate and subsequent DNA extraction as described by Arisan-Atac et al. , 1995 with the following modifications: a bead-beating step using a Fast-Prep FP 120 (Thermo Savant, Holbrook, NY) at an intensity setting of 6 for 30 sec was introduced after addition of CTAB buffer, fol¬ lowed by a phenol-chloroform-isoamyl alcohol (25:24:1) extrac¬ tion (Griffiths et al. , 2000) . The purified DNA was resuspended in 50 μl of sterile distilled water and stored at -8O0C. DNA ex¬ traction from wheat spikes was done from 3 different aliquots of each spike to compensate for variations in extraction effi¬ ciency.
In addition to the beta-tubulin sequences of 9 Austrian F. graminearum isolates (Table 1) , a total number of 191 sequences of F. graminearum and fungal species either closely related on the beta-tubulin sequence level (O'Donnell et al. , 1998) or also associated with head blight in wheat (Edwards et al., 2001) (F. graminearum [47], F. poae [75], F. sporotrichoides [36], F. pseudograminearum [18], F. cerealis [6], F. culmorum [2], F. lunulosporum [1] and G. avenacea [6]) were extracted from the NCBI database and aligned using the Vector NTI software package (InforMax Inc., Frederick, MD) . Primers were designed from the derived consensus sequence with the Primer Express® software (Ap¬ plied Biosysterns, Foster City, CA) . The primers FGtubf (5'-GGTCTCGACAGCAATGGTGTT-S') and FGtubr
(5'-GCTTGTGTTTTTCGTGGCAGT-S') specifically amplified a 111 bp fragment of the beta-tubulin gene of F. graminearum which is quantified by the TaqMan probe FGtubTM (FAM-5'ACAACGGCACCTCT- GAGCTCCAGC3' -TAMRA) . The primers and probe were analysed for specificity in-silico by blast analysis using the NCBI Blast feature (http://www.ncbi.nlm.nih.gov/BLAST) .
For the generation of a plasmid standard a 570 bp fragment of the beta-tubulin gene of F. graminearum isolate IFA 66 encom¬ passing the target region of the real-time assay was cloned into a pGEM-T Vector (Promega, Madison, WI) . After transformation of Escherichia coli JM 109 with the plasmid and purification of the plasmid DNA with the Plasmid Midi Kit (Qiagen, Hilden, Germany) , the concentration of the plasmid standard solution was measured photometrically and the standard was diluted in lOfold steps in a 10 ng/μl poly [d(I-C)] solution as unspecific DNA background (Roche Diagnostics, Mannheim, Germany) .
PCR was monitored on an iCycler iQ Real-Time Detection Sys¬ tem (Biorad, Hercules, CA) . Reaction mixtures (25 μl total volume) contained 2.5 μl of template DNA dilution, 7.5 pmol of FGtubf, 7.5 pmol of FGtubr, 6.25 pmol of FGtubTM, 12.5 μl of iQ Supermix (Biorad) and 10 μl of sterile bi-distilled water. The PCR programme was the following: 95°C for 3 min 30 sec (denatur- ation, activation of polymerase, measuring of well factors), 40 cycles of 95°C for 15 s, 67°C for 15 s and 720C for 20 s. All reactions were performed in triplicates and in at least three 10-fold dilution steps of template DNA. The number of cycles in the PCR was set to 40, as the 40th cycle represents the extra¬ polated threshold cycle for a reaction with a theoretical single copy of the template DNA. A total of six 10-fold dilution steps of plasmid standard (10-106 gene copies) were run in triplicate on every wellplate as well as a no-template control (water in¬ stead of sample) and a no-amplification control (containing plasmid standard and 0.01% SDS) . PCR efficiency was calculated from threshold cycles of these standard dilution steps. As a positive control the DNA extracts from all used isolates were also tested with the beta-tubulin PCR assay targeting all Fusarium species described in Yli-Mattila et al., 2004. GenBank accession numbers for beta-tubulin sequences obtained from 16 Austrian Fusarium isolates used in this study are included in Table 1.
Table 1
Origin* Host GenBank ac¬ Detected Detec¬
Isolate cession in gener¬ ted in number al β-tu- TaqMan bυlin assay assay
F. graminearum IFA 66 IFA durum wheat AY635186 + + kernel
F. graminearυm IFA 75 IFA maize ker¬ AY629348 + + nel
F. graminearum IFA IFA durum wheat AY629350 + + 77 .1 kernel
F. graminearum IFA 103 IFA maize ker¬ AY629342 + + nel
F. graminearum IFA 110 IFA maize ker¬ AY629343 + + nel
F. graminearum IFA 126 IFA winter whe¬ AY629344 + + at kernel
F. graminearum IFA 141 IFA soil AY629345 + +
F. graminearum IFA 165 IFA winter AY629346 + + wheat ker¬ nel
F. graminearum IFA 191 IFA maize ker¬ AY629347 + + nel
Figure imgf000013_0001
a ICE, Fusarium strain collection Institute of Chemical Engineer¬ ing, Vienna, Austria; IFA strain collection of IFA, Tulln, Aus¬ tria.
For artificial inoculation of wheat in the field experiment the IFA 191 isolate was used (F. graminearum isolated from a Triticum durum kernel in 1990 in Austria) . It was stored (at 2- 40C) in soil cultures for stable long-term storage (Smith and Onions, 1994) . Inoculum was prepared with the bubble breeding method (Mesterhazy, 1978) . A modified Czapek-Dox medium (con¬ taining/1 of bi-distilled water in g: glucose, 20; KH2PO4, 0.5; NaNO3, 2; MgSO4.7H2O, 0.5; yeast extract, 1 and 1 ml of a 1% FeSO4 solution (w/w) ) supplemented with 0.1 g streptomycin sulphate/1 and 0.01 g neomycin/1 after autoclaving was used for inoculum production. After 1 week the mycelium suspension was ready and 300 ml of the suspension was diluted in 1 1 of bi-distilled wa¬ ter for inoculation.
The wheat line E2-23-T was grown in 2 m2 plots at the IFA experimental field. This wheat line is a doubled haploid line originating from a cross between CM82036 and Remus and was pro¬ duced with the maize-wheat pollination system. It is a very sus¬ ceptible line and does not have a quantitative trait locus (QTL) for Fusarium head blight resistance on chromosome 5A and 3B (Buerstmayr et al., 2002; Buerstmayr et al., 2003) .
The wheat line was inoculated at 50% anthesis by spraying 100 ml/m2 of the inoculum suspension described above. To promote infection an automated mist irrigation system was used to apply moisture. Water was applied in 2 pulses of 10 s each at 15 min intervals during 17 hours after inoculation. Before inoculation 10 flowering spikes were cut and removed from the plot. On day 1, 2, 4, 6, 8 and 16 after inoculation the sampling procedure was repeated. All spikes were immediately stored at -8O0C after sampling for further investigations.
RESULTS:
The primers, probe and PCR protocol were evaluated by ampli¬ fying DNA from pure culture isolates of F. gram±nearum as well as the developed plasmid standard. Specificity was assessed by amplification of DNAs from various isolates which are either closely related to F. graminearum and/or also causing head blight in wheat. The β-tubulin gene of all non F. gram±nearum isolates failed to be amplified in the reaction while DNA from all isolates yielded products in the general Fusarium PCR assay (Table 1) . All PCR products were of the expected size when ex¬ amined by agarose gel electrophoresis.
All F. graminearum isolates tested could be detected with the developed method. Interestingly one additional Fusarium isolate originally included in the group of F. graminearum isol¬ ates, was presumptively identified by sequence comparison to ac¬ tually be closer related to F. flocciferum (O'Donnell et al., 1998) . Despite only one mismatch in nucleotide sequence in the binding area of the TaqMan probe this isolate was not detected by our PCR approach. The amplification of the standard dilution series yielded linear and reliable results (R2>0.997) in the range from 10 to 106 copies of the beta-tubulin gene per PCR re¬ action (range of quantitation) . The qualitative detection limit for the beta-tubulin gene was as low as 5 gene copies.
In the next step the efficiency of the amplification was in¬ vestigated using plasmid standard as well as known amounts of F. graminearum sample isolate DNA as template in the presence of unspecific DNA, such as the DNA from isolates closely related to F. graminearum (Table 1) and DNA extracted from uninfected wheat spikes. No change in threshold cycle values could be observed under these conditions as compared to amplification without a DNA background. In addition the overall efficiency of the PCR amplification was very high regardless of template origin or un¬ specific DNA background (95-98.8%) .
The developed method was applied on the total DNA extracted from spikes of infected wheat plants in all states of infection. The β-tubulin gene could not be detected in samples from unin¬ fected wheat. However, detection was possible in all samples taken after inoculation with F. graminearum isolate IFA 191. There was a distinct gradual increase in gene copies measured, corresponding to the fungal growth during the infection of the wheat spike (Fig. 1) . Detection was possible from the first day post inoculation, while first symptoms of the infection (water soaked spots) appeared 7 to 10 days after inoculation (see Fig. 1) . Data of all aliquots, dilution steps and replicates are in¬ cluded in Fig.l.
The method according to the present invention allows a fast, species-specific identification and quantitation of plant-infec¬ tions by F. graminearum at very early stages where classical mi¬ crobiological and toxin analysis methods fail to detect the pathogen (McCartney et al., 2003) . It can be applied on DNA ex¬ tracted directly from presumptively infected plant material and is not affected by an unspecific background of either plant or fungal DNA, even from other pathogens causing head blight. It allows a reliable estimation of the fungal genomes and can de¬ tect as few as 5 gene copies. The assay is cheaper and less time consuming than microbiological identification methods. The range of reliable quantification was higher than in studies published previously where the linearity was only achieved over 4 to 5 or¬ ders of magnitude (Cullen et al., 2001; Filion et al., 2003; Winton et al., 2002) .
Combined with the analysis of genes contributing to the tri- chothecene production the presented method will be an invaluable tool for the study of F. graminearum infection processes. The sensitivity of the method would even allow the quantitation of fungal growth in different plant tissues during the progress of infection (Stoyan et al., 2003) . Due to its simplicity it could also be used in routine analysis and the monitoring of F. graminearum epidemics or the monitoring of pest control meas¬ ures.
References
Arisan-Atac, I., Heidenreich, E., Kubicek, CP. , 1995. FEMS Mi¬ crobiol. Lett. 126, 249-255.
Buerstmayr, H., Lemmens, M., Hartl, L., Doldi, L., Steiner, B., Stierschneider, M., Ruckenbauer, P., 2002. Theor. Appl. Genet. 104, 84-91.
Buerstmayr, H., et al. , 2003. Theor. Appl. Genet. 107, 503-8. Cook, R.J., 1968. Phytopathology 78, 1673-1677. Cook, R.J., 1981. In: Nelson, P.E., Toussoun, T.A. (Eds.), Fusarium diseases, biology and taxonomy, The Pennsylvania State University Press, University Park, pp. 39-52.
Cullen, D.W., Lees, A.K., Toth, I.K., Duncan, J.K., 2001. Eur. J. Plant Pathol. 107, 387-398.
Edwards, S.G., Pirgozliev, S.R., Hare, M.C, Jenkinson, P., 2001. Appl Environ Microbiol 67, 1575-80.
Filion, M., St-Arnaud, M., Jabaji-Hare, S.H., 2003. J. Microbi¬ ol. Methods 53, 67-76.
Griffiths, R.I., Whiteley, A.S., O'Donnell, A.G., Bailey, M.J., 2000. Appl. Environ. Microbiol. 66, 5488-5491.
Kommedahl, T., Windels, CE. , 1981. In: Nelson, P.E., Toussoun, T.A. (Eds.), Fusarium diseases, biology and taxonomy, The Pennsylvania State University Press, University Park, pp. 94- 103.
Lee, T., Han, Y.K., Kim, K.H., Yun, S.H., Lee, Y.W., 2002. Appl. Environ. Microbiol. 68, 2148-54.
Manka, M., Visconti, A., Chelkoski, J., Bottalico, A., 1985. Phytopathol. Z. 113, 24-29.
Marasas, W.F.O., Nelson, P.E., Toussoun, T.A., 1984. Toxigenic Fusarium species: identity and mycotoxicology. The Pennsylvania State University Press, University Park
McCartney, H.A., Foster, S.J., Fraaije, B.A., Ward, E., 2003. Pest. Manag. Sci. 59, 129-142.
Mesterhazy, A., 1978. J. Phytopathol. 93, 12-25. O'Donnell, K., Cigelnik, E., Casper, H.H., 1998. Genet. Biol. 23, 57-67.
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Stoyan, R.P., Edwards, S.G., Hare, M.C, Jenkinson, P., 2003.
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Int. J. Food Microbiol., In Press.

Claims

Claims :
1. A method for the detection of Fusarium graminearum (Gibber- ella zeae) comprising the steps:
- providing a sample containing a nucleic acid,
- contacting said sample with at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises within the β-tubulin nucleic acid sequence of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence S'-RXTTTTCGTX^XSAGT-S1 (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the β-tubulin nucleic acid sequence located upstream of the hy¬ bridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid sequence SEQ ID NO 1, X1 is guanine, adenine or inosine, X2 is cytosine, adenine or in- osine, and wherein the at least one forward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer,
- subjecting the sample contacted with the at least one for¬ ward primer and the at least one reverse primer to a nucleic acid amplification technique, and
- optionally determing the presence of Fusarium graminearum (Gibberella zeae) in a sample by detecting a nucleic acid amplification product.
2. The method according to claim 1, characterised in that the at least one reverse primer and/or the at least one forward primer comprise (s) 14 to 50, preferably 16 to 45, more preferably 18 to 40, especially 20 to 30, nucleic acid residues.
3. The method according to claim 1 or 2, characterised in that the at least one reverse primer is selected from the group con¬ sisting of 5'-GCTTGTGTTTTTCGTGGCAGT-S' (SEQ ID NO 2),
5'-GCTTGTGTTTTTCGTAGCAGT-3' (SEQ ID NO 3),
5'-GCTTGTATTTTTCGTGGCAGT-3' (SEQ ID NO 4) and
5'-GCTTGTGTTTTTCGTGGAAGT-S' (SEQ ID NO 5) .
4. The method according to any one of claims 1 to 3, character¬ ised in that the nucleic acid amplification technique is a poly¬ merase chain reaction technique.
5. The method according to claim 4, characterised in that the polymerase chain reaction technique is selected from the group consisting of real-time PCR, preferably TaqMan PCR, quantitative PCR, nested PCR, assymetric PCR, multiplex PCR, inverse PCR, rapid PCR and combinations thereof.
6. The method according to any one of claims 1 to 5, character¬ ised in that the at least one forward primer hybridises within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) .
7. The method according to any one of claims 1 to 6, character¬ ised in that the at least one forward primer comprises the se¬ quence 5'-GGTCTCGACAGCAATGGTGTT-3' (SEQ ID NO 6) or
5'-GGTCTTGACAGCAATGGTGTT-3' (SEQ ID NO 7) .
8. The method according to any one of claims 1 to 7, character¬ ised in that the nucleic acid amplification technique is per¬ formed with at least one oligonucleotide probe hybridising in between of the at least one forward primer and the at least one reverse primer within the β-tubulin cluster of Fusarium gramin¬ earum (Gibberella zeae) .
9. The method according to claim 8, characterised in that the at at least one oligonucleotide probe is tagged with a dye, prefer¬ ably a fluorescent dye, and optionally with a quencher.
10. The method according to claim 8 or 9, characterised in that the at least one oligonucleotide probe comprises the sequence
5'-ACAACGGCACCTCTGAGCTCCAGC-3' (SEQ ID NO 8) or 5'-ACAACG- GTACCTCTGAGCTCCAGC-3' (SEQ ID NO 9) .
11. The method according to any one of claims 1 to 10, charac¬ terised in that the amplified nucleic acid product is addition¬ ally tagged for detection by a technique selected from the group consisting of DNA-tagging by random-priming, DNA-tagging by nick-translation, DNA-tagging by polymerase chain reaction, oli¬ gonucleotide tailing, hybridisation, tagging by kinase activity, fill-in reaction applying Klenov fragment, photobiotinylation and combinations thereof.
12. The method according to any one of claims 1 to 11, charac¬ terised in that the amplified nucleic acid product is detected by gel electrophoresis, Southern-blot, photometry, chromatogar- phy, colorimetry, fluorography, chemoluminscence, autoradio¬ graphy, detection by specific antibody and combinations thereof.
13. A kit for the detection of Fusarium graminearum (Gibberella zeae) comprising at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybrid¬ ises within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-R1TTTTCGTX1GX2AGT-S1 (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the β-tubulin nucleic acid se¬ quence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid se¬ quence SEQ ID NO 1, X1 is guanine, adenine or inosine, X2 is cytosine, adenine or inosine, and wherein the at least one for¬ ward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complementary to the at least one reverse primer.
14. The kit according to claim 13, characterised in that the at least one reverse primer and/or the at least one forward primer comprise (s) 14 to 50, preferably 16 to 45, more preferably 18 to 40, especially 20 to 30, nucleic acid residues.
15. The kit according to claim 13 or 14, characterised in that the at least one reverse primer is selected from the group con¬ sisting of 5I-GCTTGTGTTTTTCGTGGCAGT-3I (SEQ ID NO 2),
5'-GCTTGTGTTTTTCGTAGCAGT-3' (SEQ ID NO 3),
5'-GCTTGTATTTTTCGTGGCAGT-3' (SEQ ID NO 4) and
5'-GCTTGTGTTTTTCGTGGAAGT-S' (SEQ ID NO 5) .
16. The kit according to any one of claims 13 to 15, character¬ ised in that the at least one forward primer hybridises within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) .
17. The kit according to any one of claims 13 to 16, character¬ ised in that the at least one forward primer comprises the se- quence 5'-GGTCTCGACAGCAATGGTGTT-3' (SEQ ID NO 6) or 5•-GGTCT- TGACAGCAATGGTGTT-3' (SEQ ID NO 7) .
18. The kit according to any one of claims 13 to 17, character¬ ised in that the kit comprises at least one oligonucleotide probe hybridising in between of the at least one forward primer and the at least one reverse primer within the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) .
19. The kit according to claim 18, characterised in that the at at least one oligonucleotide probe is tagged with a dye, prefer¬ ably a fluorescent dye, and optionally with a quencher.
20. The kit according to claim 19 or 20, characterised in that the at least one oligonucleotide probe comprises the sequence 5I-ACAACGGCACCTCTGAGCTCCAGC-31 (SEQ ID NO 8) or 5'-ACAACG- GTACCTCTGAGCTCCAGC-3' (SEQ ID NO 9).
21. Use of at least one forward primer and at least one reverse primer, wherein the at least one reverse primer hybridises with¬ in the β-tubulin cluster of Fusarium graminearum (Gibberella zeae) and comprises the nucleic acid sequence 5'-R1TTTTCGTX1GX2AGT- 31 (SEQ ID NO 1), wherein R1 comprises at least one nucleic acid residue of the β-tubulin nucleic acid sequence located upstream of the hybridisation site of the nucleic acid sequence SEQ ID NO 1 and subsequent to the nucleic acid sequence SEQ ID NO 1, X1 is guanine, adenine or inosine, X2 is cytosine, adenine or inosine, and wherein the at least one forward primer hybridises upstream of the hybridisation site of a nucleic acid sequence complement¬ ary to the at least one reverse primer, for the detection of Fusarium graminearum (Gibberella zeae) .
PCT/EP2005/053394 2004-07-15 2005-07-14 Method for the detection of fusarium graminearum WO2006005770A2 (en)

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EP05778976A EP1766083B1 (en) 2004-07-15 2005-07-14 Method for the detection of fusarium graminearum
CA002569190A CA2569190A1 (en) 2004-07-15 2005-07-14 Method for the detection of fusarium graminearum
AT05778976T ATE508204T1 (en) 2004-07-15 2005-07-14 METHOD FOR DETECTION OF FUSARIUM GRAMINEARUM
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US20080008998A1 (en) 2008-01-10
US7919243B2 (en) 2011-04-05
DE602005027841D1 (en) 2011-06-16
CA2569190A1 (en) 2006-01-19
WO2006005770A3 (en) 2006-05-18
EP1766083B1 (en) 2011-05-04
ATE508204T1 (en) 2011-05-15

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