At this time, colonies formed by 10–15 yeast cells were selected under a stereomicroscope, picked up with a sterile toothpick and placed in 100 μL of sterile water. The cell suspension was spread on solid
potato-dextrose agar (PDA) medium and placed at 24 °C. After 4–7 days of growth, small colonies appeared (initial culture in Table 1). The fuzzy colonies were subcultured in liquid medium (10 mL of PDB) for 1 week (24 °C, 100 r.p.m.). The cell www.selleckchem.com/products/BIRB-796-(Doramapimod).html cultures were diluted to 500 cells mL−1, spread on PDA medium and cultured for 4–7 days (subculture 1 in Table 1). This procedure was repeated three times (subcultures 2 and 3 in Table 1). Strains showing 100% of fuzzy colonies after subculture 3 were defined as stable fuzzy strains and tested for their pathogenicity. Using the previous protocol, the ability of U. maydis, S. reilianum and M. penicillariae to produce solopathogenic strains was compared. For each species, teliospores from the different isolates were mixed to MG-132 ensure genetic diversity. One hundred sterile teliospores were analysed for each species. Cultures were performed on PDA medium with 0.5% charcoal for U. maydis, on PDA for S. reilianum and on both media by plate replication for M. penicillariae. The pathogenicity of the stable fuzzy strains was tested on the corresponding hosts for each species. Except for SRZS1 experiments where 40 plantlets were used, plant infection tests were carried
out on 10 plantlets for each strain. For S. reilianum, artificial inoculations were performed on maize plantlets (LMZ66; Limagrain, France) using Dynein a sterile syringe to inoculate the fungus in 10-day-old plantlets. A volume of 20 μL of cell suspension (107 cell mL−1) was injected into the stem, near the crown. Inoculated plants
were analysed 6 weeks postinoculation (w.p.i.) for PCR fungal detection on caulinar apices or for symptom observation (chlorotic spots on leaves, sorus formation). For U. maydis, foliar inoculations were performed on 3-week-old maize plantlets (LMZ66; Limagrain) and the formation of galls was observed 2 w.p.i. (Holliday, 1974). Inoculation of pearl millet (Sanyo; ICRISAT, Bamako, Mali) was carried out by infecting young floral spikelets and the symptoms were observed 5 w.p.i. (Wilson, 1995). Propidium iodide (Molecular Probes) staining was used to observe nuclei with a confocal laser scanning system (SP2 SE, Leica, Germany) equipped with an upright microscope (DM 6000, Leica). Cells from young 24-h cultures were fixed in 70% ethanol, rinsed with water, treated with RNAse A (65 °C, 10 min) and then incubated with propidium iodide, final concentration 0.1 μM, for 1 h before observation. DNA extractions were performed using the CTAB procedure (Gardes & Bruns, 1993). In planta PCR detection of S. reilianum was performed based on the amplification of the internal transcribed spacer (ITS) ribosomal regions using specific primers.