All yeast isolates were tested for antagonistic activity against A.
tubingensis (dominant species in Cyprus vineyards, unpublished
data) in a detached berry test as described previously
(Dimakopoulou et al., 2008). The yeasts were grown in liquid culture
of Yeast Malt extract (YM, containing 3 g yeast extract, 3 g
malt extract, 5 g bactopeptone and 10 g glucose per lt) in a rotary
shaker at 140 rpm for 48 h at 25 C. The yeast cells were centrifuged
at 3000 rpm for 3 min at 4 C and a suspension of cells
of each isolate was prepared (107 cells ml1) in SDW containing
0.01% Agral Syngenta (a wetting and spreading agent). Mature
grape berries of the Red Globe variety (available throughout the
year) were detached from bunches and surface-sterilized with 1%
commercial sodium hypochlorite for 5 min and rinsed three times
in SDW. Then the berries were immersed for 3 min in the yeast
suspension. Control berries were treated with SDW containing
0.01% Agral. The next day, a calibrated wound (about 2 mm diameter)
was made on each berry with a sterile needle. The wound was
spot-inoculated with 20 ll conidial suspension (104 conidia ml1)of A. tubingensis or SDW containing 0.01% Agral as negative control.
The A. tubingensis isolate was grown for 7 days on potato dextrose
agar (PDA) at 25 C and the conidial suspension was obtained
by washing plates with SDW containing 0.02% Tween-20 (Sigma–
Aldrich). The berries were incubated at 25 C for 7 days in plastic
boxes under 100% relative humidity that was maintained by the
addition of 30 ml sterile water to each box. Each yeast isolate test
consisted of fifteen berries and the experiments were repeated at
least twice. The percentage of fungal growth inhibition was determined
7 days after A. tubingensis inoculation, using the formula:
100 [(diameter of fungal colony growth on berry treated with
yeast/without yeast) 100].