Axioscope 2 microscope

Agar plugs (6 mm diam) were taken from the edge of actively growing cultures on MEA and transferred onto the centre of 9 cm diam Petri dishes containing 2 % tap water agar supplemented with sterile pine needles (PNA; Smith et al. 1996 ), potato dextrose agar (PDA), oatmeal agar (OA) and MEA (Crous et al. 2009 ), and incubated at 20–21 °C under a 12 h near-ultraviolet light/12 h dark cycle to induce sporulation as described in recent studies (Gomes et al. 2013 (link), Lombard et al. 2014 ). Colony characters and pigment production on MEA, OA and PDA were noted after 10 d. Colony colours were rated according to Rayner (1970) . Cultures were examined periodically for the development of ascomata and conidiomata. Colony diameters were measured after 7 and 10 d. The morphological characteristics were examined by mounting fungal structures in clear lactic acid and 30 measurements at ×1000 magnification were determined for each isolate using a Zeiss Axioscope 2 microscope with interference contrast (DIC) optics. Descriptions, nomenclature and illustrations of taxonomic novelties are deposited in MycoBank (www.MycoBank.org;Crous et al. 2004b ).

Axenic cultures were grown on synthetic nutrient-poor agar (SNA; Nirenburg 1981) amended with 1-cm² sterile filter paper and carnation leaf pieces, and on PDA as described by Cabral et al. (2012a) . Gross morphological characteristics were studied by mounting the fungal structures in 85 % lactic acid and 30 measurements were made for all taxonomically informative characters at ×1000 magnification using a Plan-Apochromat × 100/1.4 oil immersion lens (Carl Zeiss, Germany) mounted on a Zeiss Axioscope 2 microscope, with differential interference contrast (DIC) illumination. The 95 % confidence levels were determined for the conidial measurements with extremes given in parentheses. For all other fungal structures measured, only the extremes are provided. Colony colour was assessed using 7-d-old cultures on PDA incubated at room temperature and the colour charts of Rayner (1970) . All descriptions, illustrations and nomenclatural data were deposited in MycoBank (Crous et al. 2004 ). Optimal and cardinal growth temperatures were determined by inoculating 90 mm diam PDA plates with a 4 mm diam plug cut from the edge of an actively growing colony. Each isolate was incubated at 4, 10, 15, 20, 25, 30, and 35 °C with three replicate plates per strain at each temperature. Colony diameter of each isolate was determined after 1 wk by measuring the orthogonal directions.

Differential interference contrast (DIC) and fluorescence microscopy images were captured from living cells with Axioscope 2 microscope (Zeiss; Jena, Germany). Images were acquired with a Cool‐SNAP‐HQ 12‐bit monochrome digital camera (Roper Scientific; Trenton, NJ, USA). Yeast cultures were grown exponentially in liquid growth medium (YPD or YPG) supplemented with the indicated drugs. Cell samples were collected and concentrated 10‐fold by centrifugation (12 000 g, during 1 min at room temperature); then, cell suspensions were incubated with 1 μg ml⁻¹ 4′,6‐diamidino‐2‐phenylindole (DAPI) for 5 min to stain the DNA. More than 200 cells from at least three independent experiments were analysed in each measurement.