The fungal strains used are listed in Table . The fungal cultures were maintained at 10°C on SNA medium (0.5 g l-1 MgSO4 × 7 H2O, 1 g l-1 KNO3 (Roth, Karlsruhe, Germany), 0.2 g l-1 sucrose, 0.2 g l-1 glucose, 0.5 g l-1 KCl, 1 g l-1 KH2PO4 and 15 g l-1 agar (Merck, Darmstadt, Germany)). The Fusarium spp. cultures for DNA extraction were grown for 10 days in 100 ml Potato Dextrose Broth (PDB, 24 g l-1; Scharlau, Barcelona, Spain). The fungal mycelium was harvested by filtration and freeze-dried.
DNA isolation from pure fungal cultures grown in liquid media
We used a variant of the CTAB method [22
], simplified by others [e.g. [23
]], and modified in our laboratory as follows. The lyophilized mycelium (200 mg) was pulverized in a mortar with a small amount of sand (Riedel-de Haen, Hanover, Germany). The ground mycelium was transferred into a 50-ml centrifugation tube containing 10 ml of TES buffer (100 mM Tris (Roth, Karlsruhe, Germany), 20 mM EDTA (Merck, Darmstadt, Germany), 1% (w/v) SDS (Biomol, Hamburg, Germany), pH set to 8.0 with HCL) and 4 mg proteinase K (Merck, Darmstadt, Germany). The lysis mixture was incubated at 45°C for 45 min and the content of the tubes was mixed thoroughly by turning the tubes every 10 min. Subsequently, 3.9 ml of 5 M NaCl (Fluka, Buchs, Switzerland) were added and the sample was mixed before adding 1.4 ml of 10% (w/v) cetyltrimethylammonium bromide (CTAB, Merck, Darmstadt, Germany). The samples were incubated for 10 min at 65°C, cooled in an ice/water bath, and then 10 ml chloroform-isoamyl alcohol (24:1, Roth, Karlsruhe, Germany) were added. After mixing the emulsion thoroughly, the tubes were kept over night in an ice/water-bath. The upper phase (including a small part of the lower phase) was transferred into another centrifugation tube by pipetting and spun for 20 min at 4,000 g (5°C). The watery phase was transferred to a new tube containing 10 ml isopropanol (Roth, Karlsruhe, Germany) at room temperature, mixed thoroughly and centrifuged for 10 min at 4,000 g and room temperature. The supernatant was decanted and the pellet was rinsed with 70% (v/v) ethanol (Roth, Karlsruhe, Germany), dried and dissolved in 4.5 ml TE (10 mM Tris, 1 mM EDTA, pH set to 8.0 with HCl). In spite of the large volume of buffer used to dissolve the DNA, the process took 6 h or longer in some extractions. Dissolving the pellet can be speeded up by heating the tubes to 40°C and then mechanically destroying the pellet. Undissolved material was removed by centrifugation; the DNA was concentrated by ethanol precipitation (1/10 vol. of 5 M ammonium acetate (Merck, Darmstadt, Germany) and 2.5 vol. of 96% (v/v) ethanol) and dissolved in 0.5 ml TE. The quality and quantity of DNA were assessed by electrophoresis in 0.8% (w/v) agarose gels (Cambrex, Rockland, ME, USA) prepared in TAE buffer (40 mM Tris, 1 mM EDTA, pH set to 8.5 with acetic acid, Riedel-de Haen, Hanover, Germany). The electrophoresis was carried out at 4 V cm-1
for 60 min. Double-stranded DNA was stained with ethidium (ethidium bromide, 2 mg l-1
), (Applichem, Darmstadt, Germany). The gels were documented with the help of a digital imaging system (Vilber Lourmat, Marne la Vallee, France). Densitometry values were compared with those of Lambda Phage DNA (methylated, from Escherichia coli
host strain W3110, Sigma, Taufkirchen, Germany). The densitometry was performed using Multi Analyst-Software (BioRad, Hercules, CA, USA).
Isolation of fungi from wheat rachides
To identify the infectious agent causing the FHB, rachides of symptomatic heads were isolated and dissected into pieces spanning 2–3 segments of the spike axis. The rachis samples were rinsed with tap water and surface-sterilized by incubating for 3 min in 3% (v/v) sodium hypochlorite solution (Roth, Karlsruhe, Germany). They were then dried, inserted into agar in PDA-plates (PDB + 1.5% (w/v) agar) and incubated at 20°C for 3–5 days. The mycelia growing out of the rachides were preserved by freezing in 15% (v/v) glycerol (Roth, Karlsruhe, Germany) at -80°C.
DNA extraction from plant material
The rachides were dried at 60°C over night and a single section (2–3 segments) was ground in a ball mill (Mixer Mill MM 200, Retsch, Haan, Germany) in a stainless steel container with a 20-mm, 32-g steel sphere (Retsch, Haan, Germany) for 30 s at maximum speed. The milling container was rinsed with 1.4 ml CTAB-buffer (10 mM Tris, 20 mM EDTA, 0.02 M CTAB, 0.8 M NaCl, 0.03 M N-laurylsarcosine (Fluka, Buchs, Switzerland), 0.13 M sorbitol, 1% (w/v) polyvinylpolypyrolidone, pH set to 8.0 with NaOH (Merck, Darmstadt, Germany). Two μl mercaptoethanol (Fluka, Buchs, Switzerland) and 20 μg proteinase K (from a stock solution 20 mg/ml) were added shortly before use and the mixture was transferred to a 2-ml tube. After an initial incubation period of 10 min at 42°C and a second incubation for 10 min at 65°C, during which the content of the tubes was mixed each 3 min, 0.8 ml of chloroform-isoamyl alcohol (24:1) were added. The samples were then thoroughly emulsified and centrifuged for 10 min at 5,000 g at room temperature. 750 μl of the upper phase were transferred to a 1.5-ml tube containing 500 μl isopropanol, mixed, incubated for 20 min at room temperature and centrifuged for 15 min at 15,000 g at room temperature. The pellet was washed with 70% (v/v) ethanol, dried and dissolved in 200 μl TE. To ensure that the DNA was dissolved completely, the sediment covered by the TE buffer was incubated over night at 4°C. The quality and concentration of DNA were assessed by agarose electrophoresis as described above.
DNA extraction from the mycelia grown on agar plates
To extract the DNA from a mycelium growing on the surface of a small agar plaque, the protocol for plant material (see above) was slightly modified. At the beginning of the procedure, the agar plaque was homogenized with 1 ml CTAB-buffer in a reciprocal mill in a 2-ml tube with 9 wolfram carbide spheres (diameter 3 mm, Retsch, Haan, Germany). At the end of the procedure, the DNA was dissolved in 100 μl TE-buffer. A 1:100-dilution was used in the PCR.
The iCycler System (BioRad, Hercules, CA, USA) was used for amplification and melting curve analysis. Primers Fg16N F (ACAGATGACAAGATTCAGGCACA) and Fg16N R (TTCTTTGACATCTGTTCAACCCA) were used to amplify a 280 bp fragment specific for F. graminearum
]. Primers OPT18 F (GATGCCAGACCAAGACGAAG) and OPT18 R (GATGCCAGACGCACTAAGAT) served to multiply a 472 bp fragment specific for F. culmorum
]. All the primers were synthesized by Operon Biotechnologies (Cologne, Germany). Both primer pairs Fg16N F/R and OPT18 F/R were derived from randomly amplified genomic fragments, the function of target sequences is unknown.
The amplification mix consisted of 1× NH4-reaction buffer (diluted from 10× NH4-reaction buffer: 160 mM (NH4)2SO4, 670 mM Tris-HCl, 0.1% (v/v) Tween-20, pH 8.8 at 25°C, Bioline, Luckenwalde, Germany), 5 mM MgCl2 (Bioline, Luckenwalde, Germany), 0.2 mM of each dATP, dTTP, dCTP and dGTP (Bioline, Luckenwalde, Germany), 0.3 μM of each primer, 0.7 u BIOTaq DNA polymerase (Bioline, Luckenwalde, Germany), 10 nM Fluorescein (BioRad, Hercules, CA, USA, diluted from 1 μM in 1× NH4-reaction buffer, to collect well factors, specific for the iCycler), 0.4× SYBR Green I solution (Invitrogen, Karlsruhe, Germany), 1 μl of template DNA and ddH2O up to 25 μl. The detection of amplification products, based on the fluorescence of SYBR Green I, was performed with filters set at 490 ± 10 nm for excitation and 530 ± 15 nm for emission.
The PCR was performed with the following cycling protocol. Initial denaturation for 1.5 min at 95°C (the denaturation time is used by the thermocycler to collect data for the calculation of the well correction factors, which is needed to compensate for differences among wells of the microtitre plate) was followed by 35 cycles with 30 s at 94°C, 45 s at 64°C, and 45 s at 72°C. The final elongation was performed for 5 min at 72°C. During the PCR, the detection of fluorescence was carried out in the annealing step of each cycle. Following amplification, the melting curves were acquired by heating the samples to 95°C for 1 min, cooling to 55°C for one min and then slowly increasing the temperature from 65°C to 95°C at the rate of 0.5°C 30 s-1, with a continuous measurement of the fluorescence.
When an electrophoretic analysis of PCR products was necessary, 4 μl of the reaction mixture were combined with 2 μl of loading buffer (100 mM EDTA, 50% (v/v) Glycerol, 0.025% (w/v) bromphenol-blue, Merck, Darmstadt, Germany) and loaded on a 1.7% (w/v) agarose gel prepared in TAE buffer. The electrophoresis and the documentation were carried out as described above.
Sample identifiers, CT
values (threshold cycles) and the results of the melting curve analysis were exported from the thermocycler into the pre-formatted Excel spreadsheets, which facilitated the automatic data processing and reporting (see Additional file 1
). The Excel file can be downloaded from [21
], its structure and function is described in detail in the Data Processing paragraph of the Results section.