Fifty-five isolates of M. graminicola and other fungal species used in the current study are listed in Table 1. Isolates used for DNA isolation were grown on yeast glucose broth (YG; 1% yeast extract, 3% glucose) at 18 °C by shaking for five days on an orbital shaker at a speed of 120 rpm. Spores were collected from YG by centrifugation and washed three times with sterile water and subsequently once with 0.6 M MgSO4 (pH 5.8). For storage for three-to-six months, the M. graminicola isolates were cultured on Malt Yeast Agar (MYA) plates then stored at 4 °C or −80 °C in PD broth medium supplemented with 10% glycerol [14].

Seeds of cultivar Ritmo (a highly susceptible wheat cultivar) were germinated on filter paper in the dark at 25 °C. After 24 h, the seeds were placed at 5 °C for 48 h followed by incubation at 25 °C for 24 h. The plants were then transferred to a growth chamber at 19 °C, with a day/night regime of 16 h/8 h. Wheat seedlings were inoculated by placing 10 μL of M. graminicola pycnidiospore suspension (containing 10⁶ spores) on each emerged leaf (leaf 2). Control plants were treated with distilled water. The inoculated plants were kept in a mist chamber for 72 h and then returned to the growth chamber at 21 °C (day)/16 °C (night) temperatures with a day/night regime of 16 h/8 h. Samples for PCR analysis were collected at 0, 2, 6, 8, 10, 12, 14, 16, 18 and 20 days after inoculation. DNA was extracted from wheat leaves according to procedures described by Guo et al. [15].

A modification of the traditional sodium dodecyl sulfate (SDS) extraction procedure was adopted [1]. Fungal mats (100 mg) were harvested and homogenized in 400 μL of sterile salt homogenizing buffer (200 mM Tris-HCl, pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). Then, 6 μL of RNase A (final concentration 20 mg mL⁻¹) was added and mixed. The samples were incubated at 65 °C for 10 min, after which 130 μL of 3 M sodium acetate, pH 5.2, was added to each sample. Samples were mixed in a Vortex for 30 s at maximum speed, and incubated at −20 °C for 10 min. The lysate was centrifuged at 13,000 rpm at 4 °C for 15 min. The supernatant was transferred to fresh tubes. An equal volume of isopropanol was added to each sample, mixed, and samples were incubated at −20 °C for 10 min. Samples were then centrifuged for 20 min at 4 °C, at 6,000 rpm. DNA pellets were washed twice with 700 μL of washing solution (100% and 70% ethanol, respectively). The DNA pellets were subsequently air-dried in an oven at 40 °C for at least 10 min. The resultant DNA pellet was then resuspended in 100 μL of 1 × TE (10 mM Tris-HCl, 1 mM EDTA) buffer, pH 8.0.

Microsatellites of varying length and type (di or trinucleotide) were selected from those identified by Karaoglu et al. [25], which are listed at http://www.mmrl.med.usyd.edu.au/an_contig.html. Microsatellite dinucleotide specific-primers were designed [(CTC TCT CTC TCT CT) G] based on microsatellite repeat sequence selected from the finished genome sequence of isolate IPO323 of M. graminicola (http://genome.jgi-psf.org/Mycgr3/Mycgr3.home.html).

PCR mixtures contained 2 mM deoxynucleoside triphosphates, 10 pmol concentrations of primer; 0.2 U of Taq polymerase (Biolab, England), 1 × buffer (Promega, Mannheim, Germany), 1.5 mM MgCl₂ and 50 ng of template DNA in a total volume of 25 μL. The reactions were carried out in a PTC-200 Thermocycler (MJ Research, Waltham, USA) as follows: 1 cycle of 2 min at 94 °C followed by 40 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 90 s and extension at 72 °C for 2 min. The thermal profile ended with a final extension at 72 °C for 7 min. All PCR amplification products were analyzed by agarose gel (1.5%) electrophoresis [26].

Real-time PCR reactions were carried out with a LightCycler instrument using the QuantiTect™ SYBR^® Green PCR Kit (Qiagen, Hilden, Germany) with 0.3 μM of each primer, 5 mM MgCl₂ and 5 μL of DNA template. DNA was replaced by sterile water in the negative control. The program used for real time PCR was 10 min at 95 °C, followed by 35 cycles of 15 s denaturation at 95 °C, 30 s annealing at 60 °C, 30 s elongation at 72 °C. Samples were placed into a glass capillary, capped, centrifuged for a few seconds in a micro-centrifuge using appropriate adapters, and then placed into the LightCycler rotor. In addition, the PCR products were recovered from the capillaries and analyzed by agarose gel (1.5%) electrophoresis and stained with ethidium bromide [27].

The specificities of the gene-specific primers designed in this study were tested in PCR amplifications using purified genomic DNA from various fungal species as template. To assess the sensitivity of the detection of M. graminicola using the designed primers, DNA dilution series containing 10 ng to 50 fg of DNA from selected isolates were subjected to conventional, and real-time PCR analyses, respectively.

The specificity of the real-time PCR assay was tested with template DNA extracted from the 55 isolates of M. graminicola and other fungal species that are listed in Table 1. Dinucleotide specific-primer amplified a single fragment from total genomic DNA of M. graminicola (Figure 1) and its inoculated leaves. There was no amplification obtained in healthy plants (HP) or the other fungal pathogens tested (Stagonospora nodorum (SN), Pseudocercosporella herpotrichoides (PT)) (Figure 2). The (CT) 7 G primer amplified a unique DNA fragment of approximately 570 bp from M. graminicola, whereas no amplification was achieved with DNA isolated from other fungal species. Using DNA extracted from M. graminicola cultures, the detection limit was 100 pg/μL with conventional PCR, whereas the detection limit was 50 fg/μL by real-time PCR, thus the real-time PCR assay was 20-fold more sensitive than conventional PCR (Figure 3).

In the assay, SYBR Green I was used as the fluorescent dye enabling real-time detection of PCR products. Characterization of the amplicons was achieved by melting point analysis 84 ± 0.2 °C. Nonspecific products such as primer dimers could readily be distinguished from PCR products by their lower melting points. PCR reactions performed on 50 ng of M. graminicola DNA with primers targeted at fungal sequences led to similar Ct and dissociation curves compared to a control devoid of DNA matrix, showing the specificity of fungal quantification (Figure 2). The system enables a 35-cycle PCR with 35 samples to be completed in 45 min, including quantification and identification of the product. Typical results from the probe melt curve analysis are shown in Figure 4. All products were subjected to analytical gel electrophoresis to confirm the 570 bp amplicon size.

Artificially M. graminicola-inoculated asymptomatic wheat leaves were collected and fungal DNA was detected with conventional PCR and real-time PCR 0, 2, 6, 8, 10, 12, 14, 16, 18 and 20 days post inoculation. Inoculated wheat leaves showed the 750-bp fragment that is diagnostic for M. graminicola infections after PCR with dinucleotide specific primers. No PCR products were generated with DNA isolated from Fusarium graminearum-inoculated or Pseudocercosporella herpotrichoides-inoculated wheat plants or from DNA isolated from healthy wheat tissue. A clear single DNA fragment was amplified from the samples taken on day 0, 2, 6, 8, 10, 12, 14, 16, 18 and 20 after inoculation (Figure 5, lanes 1–10). From inoculated wheat leaves harvested at day 0, although thoroughly washed with water prior to PCR, a clear fragment was still present representing the inoculated fungus. The intensity of the amplified DNA fragment increased significantly at 10 days after inoculation. Between 10 and 20 days after inoculation, the intensity of the amplified fragment increased more than five-fold. As mentioned before, the dinucleotide-specific primers can detect the pathogen from 10 pg of DNA isolated from infected leaf tissue in conventional PCR assays and from 50 fg in real-time PCR assays; the higher sensitivity offered by the real-time PCR assay makes it more reliable for detecting the fungus during the latent stage of infection. The sensitivity of the method would allow the quantification of fungal growth in different plant tissues during the progress of infection.