100 Y. M. Hokama, D. C. Savi, B. Assad et al.
g of glucose, and 1000 mL distillated water) under agitation for 28 days (120
rpm, 28°C). After fermentation, mycelia were separated from the fermented
liquid by filtration through Whatman nº4 filter paper. Metabolites were
extracted with ethyl acetate (3 × v) to obtain the liquid fraction, and the
mycelia were extracted using another 30 mL of ethyl acetate (Merck,
Germany, PA). The solvent was evaporated using a rotaevaporator at 45°C.
The final extracts were diluted in methanol at a concentration of 10 mg/mL.
Extract Activity against the Phytopathogen
Phyllosticta citricarpa
Antagonistic activity of the endophyte extracts against Phyllosticta
citricarpa was evaluated by two approaches: determining the growth of P.
citricarpa and inhibition of pycnidia formation.
Growth Inhibition Analysis
The inhibition of mycelial growth was evaluated by measuring colony
diameters on PDA medium, where one central disc with the phytopathogen (12
mm) was deposited in the center of each petri dish, and its growth on the plate
surface was compared with that of the phytopathogen treated with 1 mg of
extract. Measurements were recorded after 14, 21, and 28 days of incubation,
with 10 experimental replicates. Glyphosate 1.0 mg/mL (Bayer Leverkusen)
was used as a positive control.
Pycnidia Formation Analysis
To analyze pycnidia formation, Citrus limonia Osbeck leaves were
washed in water, cut into fragments (10 mm), and autoclaved in distilled
water. Three leaf fragments were placed in petri dishes with water-agar (1.5%
w/v) culture medium. Four 2-mm-thick discs of Phyllosticta citricarpa
mycelia were inoculated close to each leaf fragment, 100 μg of extract was
added to each dish, except for that of the control, in which just the
phytopathogen was cultured. The petri dishes were sealed and maintained
at 28°C with a 12-h photoperiod for 21 days. After this period, the pycnidia of
P. citricarpa that formed above the leaves were counted under a