© Benaki Phytopathological Institute
Gkounti
et al
.
48
ve plants,
Agave americana
L., in the ur-
ban landscape area of Ardittos hill (Athens,
Greece). The infestation of the agave plants
was spotted in the summer of 2012 and no
action for controlling the pest was taken pri-
or to the collection of the weevils. The adults
were collected by hand at site, while whole
plants were cut from the basis and taken to
the laboratory for the isolation of 4
th
instar
larvae. The adults were kept in polyester
cages (30 x 30 x 30cm) and the larvae in plas-
tic boxes (20 x 20 x 20cm), with an opening
covered by mesh for ventilation. All insects
were kept at controlled laboratory condi-
tions (25±1°C, 65±5% R.H. and 14:10 L:D) and
fed on a natural diet (agave slices; twice per
week) until they were used in the bioassays.
Bioassays
The application of the spore suspen-
sions was achieved through direct cuticle
contact. Groups of five, 4
th
instar larvae and
adults were immersed in the prepared aque-
ous suspensions for 30 sec after which each
group of insects was transferred in 1L rearing
boxes and provided with fresh natural diet.
The treatment was repeated 15 times (rep-
licates) for each fungus strain and concen-
tration per life stage. Furthermore, a control
treatment was included for both adults and
larvae, where insects were immersed into
an aqueous solution of 0.2% Tween 80. All
bioassays were carried out in controlled en-
vironment conditions (25±1°C, 65±5% R.H.
and 14:10 L:D). The mortality was recorded
daily for up to 11 and 21 days for larvae and
adults, respectively. When a weevil died, the
cadaver was transferred individually to a Pe-
tri dish lined with a moistened sterile filter
paper to ascertain the involvement of the
entomopathogens to the death of weevils.
The Petri dishes were sealed with parafilm
and kept at room temperature, in darkness,
to monitor for external signs of fungal infec-
tion.
Statistical analyses
General Linear Model (GLM) with a bino-
mial distribution was used to obtain Anal-
ysis of Deviance for a level of significance
α=0.05. The analysis included life stage (lar-
val and adult), species of fungi (
B. bassiana
,
I. fumosorosea
and
M. anisopliae
), and con-
centration of spore suspensions (10
6
and
10
7
spores/ml) as variables. Additionally,
the average survival times were calculated
and compared by the Kaplan-Meier surviv-
al analysis (Kaplan and Meier, 1958). All the
GLM procedures and survival analysis were
carried out in R (v. 2.1; R Foundation for Sta-
tistical Computing, Vienna, AT).
Results
The examined strains of entomopathogenic
fungi caused different levels of mortality to
S. acupunctatus
. The mean mortality of wee-
vils differed significantly at different life stag-
es, and fungus species, but it did not differ
between different concentrations of spore
suspensions (
X
2
=22.939,
df
=1,
P
<0.0001;
X
2
=9.5758,
df
=2,
P
<0.0001 and
X
2
=2.5984,
df
=1,
P
=0.1070, respectively). No mortali-
ty was recorded for adult weevils or larvae
when treated with the aqueous solution of
0.2% Tween 80 (i.e. control treatment). Inter-
actions among the variables did not affect
the mortality levels, thus, they were omitted
from the initial model.
All treatments with fungi resulted in al-
most all concentrations at 100%mortality of
larvae (Figure1a).Mortality inadultsdepend-
ed on fungus species but not on the concen-
tration of the suspensions (
X
2
=11.4223,
df
=2,
P
=0.0033 and
X
2
=2.9638,
df
=1,
P
=0.0851, re-
spectively). The treatment with
B. bassiana
at the concentration of 2.12 x 10
7
spores/ml
caused the highest mortality to adult wee-
vils (86.67±12%) compared to
M. anisopliae
and
I. fumosorosea
treatments (Figure 1b).
The lowest mortality levels to adults were
caused by
M. anisopliae
, when applied at the
low spore concentration (26.67±15.8%).
The examined strains of
B. bassiana, M.
anisopliae
and
I. fumosorosea
developed ex-
ternal fungal growth and sporulated on all
treated cadavers of both adults (Figure 2)
and larvae, regardless the spore concentra-
tion.
