fungal – sensitive species considered. For example, for the biological
system used (H. fasciculare – S. cerevisiae), reproductive
inhibition zones are well defined and distinctive in the selected
conditions. Antimicrobial potential for K. marxianus (AP = 1.50) and
C. tropicalis (AP = 1.41) were slightly different and both smaller than
the value obtained for the model yeast (AP = 2.50), which could
indicate species/strain specificities. Nevertheless, the assay proved
to be effective in what concerns the evaluation of fungal activity
against other yeast species.
The results obtained during assay optimization suggested that
the detection of fungal antimicrobial activity strongly depends
on the physiological status and growing conditions of the tested
fungus. Not only the assayed medium and its pH affected the
antimicrobial activity detected, but also the fungal incubation
period and temperature led to differences in AP values. Enlarged
inhibition halos and higher AP values were detected with increasing
fungal growth, as expected. However, whereas fungal growth
seems to gradually slow down with longer incubation periods the
halo size continues to increase with the fungal incubation period.
Most probably, more antagonistic substance is being produced
and/or accumulated over time.
The production of antimicrobial compounds might also be
related to the temperature of fungal growth. When H. fasciculare
was cultured for 6 days at increasing temperatures before
the antimicrobial assay, the growth optimal temperature was confirmed
to be 25 ◦C but the best assay condition for achieving the
highest fungal antimicrobial potential was at 30 ◦C. Any temperature
increase above 25 ◦C led to a reduction of fungal areas and
even to fungal death at 35 ◦C and 40 ◦C. Clear differences were also
detected in the optimal conditions for achieving maximal mycelia
growth and bioactivity in other fungi (Frisvad and Samson 1991;
Miao et al. 2006).
Altogether, the results show that the inhibition of yeast growth
is more noticeable when any environmental factor (media composition,
pH value, temperature) limits the growth of the filamentous
fungus. The production of antimicrobial compounds is a wellknown
strategy of plant response to different stress conditions
(Mert-Türk 2002). Also in fungi, the culture conditions have a major
impact on growth and antimicrobial compounds production (Miao
et al. 2006). The production of such bioactive substances could
then be optimized by adjusting several physical (temperature, pH
value) or chemical (media components) factors (Calvo et al. 2002;
Llorens et al. 2004). Therefore, the study of fungal antimicrobial
activity dynamics is important for establishing the best conditions
for achieving the maximal fungal growth rates and maximal antimicrobial
activity. Frequently, the conditions that allow fast fungal
growth can be unfavourable to metabolite production (Miao et al.
2006). This bioassay can be used for such purposes: to study the
dynamics of antimicrobial compounds production by filamentous
fungi.
To the best of our knowledge, this is the first time that an assay
to detect filamentous fungi antimicrobial activity is performed
directly with the antagonist fungus and a yeast as indicator sensitive
strain. This kind of assay has the great advantage of being
less time consuming and allows the detection of antimicrobial
properties using the fungus directly, without prior isolation of the
active substance(s). This is particularly useful when performing
large screenings for fungal antimicrobial activity, in order to identify
or select possible promising fungi. The susceptibility showed
by C. tropicalis to the fungus under study seems also an interesting
result regarding the control of Candida-associated opportunistic
infections, an emergent health concern