Page 16 - Volume 2,Issue 2, July 2009

© Benaki Phytopathological Institute
Albano et al. 60
gicide was kept in the freezer (-16º C) until
it was placed in the dispensers. The prepara-
tion of T. harzianum used in the experiments
contained a 9.8 x 10 6 Colony Forming Units
(CFU)/g formulation.
2.1.1. Inoculum on pollinators
For the honey bee assay, a sample of
30 honey bees exiting from hives-mount-
ed dispensers containing T. harzianum was
collected. These captures were performed
about one hour after the filling. In the bum-
ble bee assay a sample of 10 bumble bees
exiting from the hives-mounted dispensers
with T. harzianum was collected in the fol-
lowing three hours after the filling.
In both experiments, a sample of 30 pol-
linator foragers visiting the strawberry flow-
ers was also collected. During the honey
bee assay, there were no other beehives in-
stalled at a radius of 3 to 4 km. The forag-
ing bee samples collected in the experimen-
tal field thus most probably belonged to the
hives-mounted dispensers.
2.1.2. Inoculum deposited on flowers
During each assay (honey bee and bum-
ble bee), a total of 120 flower buds were
randomly marked in two groups: 60 flow-
er buds were isolated with a small tulle bag
to exclude all insect visits during the exper-
iment (control flowers); the other 60 flower
buds were kept unscreened to bee visits. For
this second group, every morning the flow-
er buds were checked, and the newly open
flowers were left exposed to the bees for
that day and collected at the end of it.
Additionaly to these two groups of flow-
ers, a third group was defined in each of the
assays: flowers exposed to a single visit. For
that, in a given location of the field and of
the greenhouse, a large area of the straw-
berry plants was kept screened with a mesh
cloth until experimental observations were
performed. During the observation periods,
the screens were removed and the open vir-
gin flowers were exposed to a single visit. In
the honey bee and bumble bee assays, sin-
gle bee visits were recorded for 20 and 30
flowers, respectively.
For the bumble bee assay, when an open
virgin flower was visited, the arrival and de-
parture of the visiting bee was vocally reg-
istered with a tape recorder for a later de-
termination of the length of each visit. After
a sample of 30 flowers (mentioned above),
the following 12 observed flowers were al-
lowed to be visited two or three times each,
performing a total of 42 observed flowers.
Whenever possible, the bumble bee that
made the corresponding visit was captured,
in a total of 30 insects.
2.2. At the laboratory
The insect and flower samples collected
during each assay were individually stored
in a glass container and carried to the labo-
ratory. All samples were kept preserved in a
freezer (-16º C) before their analysis.
To each sample collected during field-
work (flowers and insects) 20 ml of distilled
water with Tween (0.01%) were added and
the resulting suspension was shaken for
one hour. Subsequently, 400μl of the origi-
nal suspension were plated on a Trichoder-
ma selective medium. This growth medium,
modified from Smith et al. (28), contained
the following chemical compounds (g/L):
Ca(NO3)2, 1.0; KNO3, 0.26; MgSO4 . 7H2O, 0.26;
KH2PO4, 0.12; CaCl2.2H 2O, 1.0; citric acid, 0.05;
sucrose, 2.0; agar, 20.0. After adjusting the
pH to 4.5 and when temperature decreased
to 45ºC, the following antibiotics and fun-
gicides were added (g/L): tetracycline, 0.05;
streptomycine, 0.10; Senator® (70%) (oxy-
chloride + maneb + sulphur), 0.0143 and
quintozene (75%), 0.02666. Three Petri dish-
es (replicates) were used for each sample.
No dilutions were performed with the ex-
ception of the samples of bumble bees that
were captured while exiting the hives.
The Petri dishes were kept in the labora-
tory at 22ºC and the number of colonies that
developed was counted about eight days af-
ter plating took place.
2.3. Data analysis
For all the samples (flowers and insects)
that resulted from the two experiments, the
mean number of CFU was estimated from