Fluorescence brightener 28

Cell length measurements were performed on the three-first subapical cells of 7-day-old protonema grown in BCD media, stained with 10 mg. ml^-1 of Caclcofluor, (Fluorescence brightener 28, Sigma-Aldrich). Calcofluor fluorescence was imaged with a UV filter set. For cell growth rate measurements, protonemal tissue was grown on Petri dishes (30-mm diameter) on BCD media for 7-days. Images of apical chloronemal or caulonemal cells were acquired every 10-15 min during a 2-3-h period. Protonemata cell images were taken with an Olympus BX-61 microscope equipped with a color camera (Olympus DP71). The measurements of cell length and growth rate were made using FIJI-IMAGEJ software.

All in silico calculations were performed using a Dell precision T7600 Minitower workstation with two Intel^®Xeon^® processors CPU E5-268W 0, 3.1 GHz, 8 GB RAM running on the Red Hat Enterprise Linux 5 operating system. The optical brightener (OB) compounds DAST (462268 Sigma-Aldrich, EE.UU., St. Louis, MO, USA), Fluorescence brightener #28 (F3543 Sigma-Aldrich, EE.UU., St. Louis, MO, USA), and Fluorescence brightener #71 (Toronto Research Chemicals, Toronto, ON, Canada) were purchased from the indicated companies. The stock solution for each OB (1000 µM) and subsequent dilutions were prepared using K-medium (KCl, NaCl, Millli-Q Water, Darmstadt, Germany) [23 (link)].

The 200 μL of Fluorescence Brightener 28 (F3543-1G, Sigma Aldrich, Steniheim, Germany) fluorescent dye was added to 1 mg of the EPS precipitates and incubated in the dark for 25 min. After this time, the dye solution was separated from the EPS by centrifugation, and then the pellet was applied to a slide and observed with excitation at 365 nm and emission at 435 nm under an Axivert 200M confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a Zeiss LSM5 Pascal head

Fungal growth on leaf surfaces of inoculated plants treated with water alone, water amended with 1% ethanol and 0.15 % Cantor or with M1, M2 and GABA molecules, was monitored at 1 and 5 dpi using Fluorescence Brightener 28 (Calcofluor, Sigma Aldrich) staining, according to Siah et al.^{1 (link)}. Third-leaf segments (4 cm) were collected from plants of each condition before being stained with a solution of 0.1 % Calcofluor 0.1 M Tris-HCl buffer at pH 8.5 and washed with sterile distilled water. After drying in darkness at laboratory temperature, leaf segments were placed on a glass slide, covered with a cover slip and observed under a microscope with UV illumination (Nikon, Eclipse 80i). The effect of different treatments on hyphal growth was assessed at 1 dpi by determining the percentage of germinated spores, and at 5 dpi by recording four fungal cytological event classes (class 1, non-germinated spore; class 2, geminated spore with minor germ tube; class 3, geminated spore with developed germ tube; class 4, geminated spore with a strongly developed germ tube). Microscopic observations at both 1 and 5 dpi were performed on 100 randomly-chosen fungal spores on each leaf segment. Three third-leaf segments were as replicates for each condition.