Axiocam 506 color microscope

Cells were stained with NucSpot^Ⓡ Live Cell Nuclear Stains (#40082, BIOTIUM, Fremont, CA). Washings were with phosphate buffered saline (PBS) before plating in RPMI 1640 media supplemented with 10% FBS with antimycotics and antibiotics. Cells were maintained in the microscope chamber at 37°C and 5% CO₂. Stained cells were scored for mitotic figures that were bipolar, or those having ring-like chromosomal structures or multipolarity division using a ZEISS Axiocam 506 color microscope and the Live-Cell Imaging system (Carl Zeiss Microscopy LLC, White Plains, NY).

Morphological characterization of colonies, such as colony appearance, color and spore production were observed and recorded following the method of previous studies [5 (link),11 (link),16 (link)] on three media (PDA, malt extract agar (MEA, malt extract 20 g, agar 18 g, add water to 1 L) and synthetic nutrient-poor agar (SNA, monopotassium phosphate 1 g, potassium nitrate 1 g, Magnesium sulfate heptahydrate 0.5 g, potassium chloride 0.5 g, glucose 0.2 g, saccharose 0.2 g, agar 20 g, add water to 1 L)) with each isolate three replicates. Microscopic characteristics were recorded based on 20 paraphyses, 20 conidiogenous cells and 50 conidia on PDA at 25 °C in darkness. Photographs were taken from material mounted in lactic acid with Axiocam 506 color microscope (Carl Zeiss, Aalen, Germany) using Zeiss Imager Z2 software. The new species were established based on the guidelines outlined by Jeewon and Hyde [17 (link)].

For histochemical study, transcriptional reporter line pAtCLCf::GUS was generated in WT background. To carry out GUS staining assays, cold stratified seeds were sown on MS plates. One-week-old, untreated, and salt treated (100 mM NaCl for 6 h) seedlings as well as four-week-old seedling tissues were stained in the GUS staining solution [0.1 M sodium phosphate buffer pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 2 mM 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc)] for 5 min followed by overnight incubation in the dark at 37 °C without shaking. After removing staining solution several washes with 50 % ethanol were performed to remove chlorophyll until the tissues were cleared^{66 (link)}. The images of stained whole seedlings as well as various tissue parts were recorded using an Axiocam 506 color microscope (Zeiss). GUS expression in different parts of seedlings was quantified based on the relative intensities of blue coloration using ImageJ software (https://imagej.nih.gov/ij/download.html). Data presented are mean ± SE of three biological replicates, each biological replicate consisting of at least six plants. Statistical significance was determined by unpaired Student’s t test (two-tailed).