Evans blue catalog e2129

Light microscopy was used to study the in vitro interactions between Fg and the bacterial strains. For this, microscope slides were coated with a thin layer of 1 mL Potato Dextrose Agar (PDA) and allowed to solidify. 50 µL of Fg mycelia, grown for 72 h in Potato Dextrose Broth (PDB) at 25°C at 120 rpm, was applied in the center of the slide. On the adjacent side of this, 50 µL of each bacterial strain, cultured in LB liquid (pH 7.2) by incubating at 30°C at 200 rpm for 48 h, were applied. Likewise, LB broth was placed on the left side as a negative control. These slides were placed in closed Petri dishes and incubated at 25°C for 24 h. The positive control included Proline fungicide (1:10 v/v with ddH₂O). The slides were then stained with the vitality stain, Evans blue (Catalog # E2129, Sigma Aldrich, Canada) by placing 1 mL of stain on the slide, which was then incubated for 5 min at room temperature, and washed 3-4 times with deionized water. Slides were prepared in triplicate for each treatment, visualized under a light microscope and pictures were captured.

To determine whether pollen-associated bacteria had direct fungicidal activity against Fusarium isolate FgMT#1, Fusarium mycelia was placed in the center of each microscopic slide, along with candidate pollen bacterial culture to one side of Fusarium, and LB buffer to the opposite side as the negative control. After 24 h of incubation at 25˚C, the slides were stained with the vitality stain Evans blue (Catalog # E2129, Sigma Aldrich®, Missouri, USA) and imaged using a light microscope. There were 3 replicates for each bacterial candidate, with each slide placed in a separate Petri dish. See Supplementary Methods for details.