Yeasts were individually picked onto Petri dishes with PDA
medium in the form of two parallel lines 1 cm apart from each other.
A C. gloeosporioides mycelium disk of 6 mm in diameter, cultured in
PDA for 10 d, was placed at the center of the plate precisely between
the two yeast strains streak spots. The plates were incubated
at 28 ◦C until an interaction between the phytopathogen hyphae
and the test yeast (4 d of incubation) was reached. Subsequently,
samples of approximately 1 cm2 containing both organisms were
extracted with a scalpel, washed twice with distilled water and prepared
for SEM imaging, according to Bozzola and Russell (1999).
The positive control was a mycelium portion (1 cm2) taken from a
C. gloeosporioides culture, which was grown on PDA at 28 ◦C for 10 d.
The samples were placed in a modified Karnovsky fixative for 48 h
and post-fixed in 1% osmium tetroxide in sodium cacodylate buffer
(0.1 M, pH 7.2) for 1 h. Afterwards, the samples were dehydrated
by immersion in crescent ethanol series (30%, 50%, 70% and 90%)
for 10 min. Next, the samples were immersed in 3 pure ethanol
baths of 10 min each and taken to a critical point dryer (Emitech
K850), coated with gold sputter coating (Emitech K550) and examined
with a SEM (Zeiss DSM 940 A) using an acceleration voltage of
15 kV.