© Benaki Phytopathological Institute
Pathogenicity of entomopathogenic fungi on sisal weevil
47
site of origin (Velazquez
et al.,
2008), these
arthropods are not found in the invaded re-
gions. Entomopathogenic fungi and nema-
todes have been reported as potential bio-
logical control agents of the weevil (Hueso
et al.
, 2005; Velazquez
et al.,
2008). Both the
fungi and the nematodes are of great im-
portance for the biological control of
S. acu-
punctatus
, as they are able to overcome the
barrier of the concealed environment in
which the weevil develops. While the nem-
atodes are able to move within the agave
leaves and are easily able to reach the con-
cealed pest (Dembilio
et al.,
2010a), the en-
tomopathogenic fungi infect their hosts
by direct contact and subsequent penetra-
tion of their cuticle (Dembilio
et al.,
2010b).
This makes insects susceptible both to di-
rect contact with the pathogen, but also to
passive, horizontal transmission, between
healthy and infected individuals vector-
ing spores (Klein and Lacey, 1999; Quesada-
Moraga
et al.
, 2004).
It has been widely accepted that ento-
mopathogenic fungi show high intra-spe-
cific diversity, which results in different lev-
els of pathogenicity among different strains
of the same species (Ferron, 1978). Further-
more, the virulence of a specific strain to
specific insect genera has been proved to
depend upon the host insect identity from
which it was isolated (Fargues and Robert,
1983; Maurer
et al
., 1997). This is attributed
to the strong selective pressure of the host
insect environment on the pathogen (Roy
et al.,
2006) through which the origin of the
entomopathogens could affect their patho-
genicity against pests (Cherry
et al.,
2005).
Despite the importance of alternative
methods for the control of the sisal weevil,
research on the evaluation of potential bio-
logical control agents of the pest is very lim-
ited (Molina-Ochoa
et al.,
2004; Valdes Estra-
da
et al.,
2014). In the present study we aim
to evaluate the virulence of locally isolat-
ed strains of
Beauveria bassiana
(Balsamo),
Metarhizium anisopliae
(Metchnikoff) So-
rokin and
Isaria fumosorosea
(Wize) to adults
and larvae of the introduced weevil
S. acu-
punctatus
.
It is expected that the use of in-
digenous isolates of fungal entomopatho-
gens is advantageous, as they are already
adapted in specific environmental condi-
tions and could be part of a conservation bi-
ological control strategy.
Material and Methods
Entomopathogenic fungi
All entomopathogenic fungi strains used
in the laboratory assays were obtained from
the entomopathogenic fungi collection of
the Benaki Phytopathological Institute. The
strain of
B. bassiana
IBB 010 was initially iso-
lated from a
Rhynchophorus ferrugineus
(Ol-
ivier) cadaver, found in Ellinikon region
(Athens, Greece). Both strains IBB020 of
I. fu-
mosorosea
and TMB04 of
M. anisopliae
were
isolated from soil samples with the
Galleria
bait method (Zimmerman, 1986) from the
Agios Stefanos and Marathon regions (At-
tica, Greece), respectively. The molecular
identification of the strains of
I. fumosorosea
and
M. anisopliae
was previously conduct-
ed by Beris
et al.
(2013). The fungal isolations
were cultured on Sabouraud dextrose agar
(SDA) at 25°C, under dark regulated condi-
tions. Spore (conidia) suspensions were pre-
pared by scraping the surface of 2 week old
cultures into an aqueous solution of 0.2%
Tween 80, after which the suspensions were
vortexed for 60 s and filtered twice using a
sterile nylon membrane to remove myceli-
um. Subsequently, the concentration of the
spore suspensions was determined by the
use of a standard haemocytometer. The
concentrations of spore suspensions used
in bioassays were 2.12 x 10
7
and 2.12 x 10
6
spores/ml for
B. bassiana
, 1.27 x 10
7
and 1.27
x 10
6
spores/ml for
M. anisopliae
and 7.45 x
10
6
spores/ml for
I. fumosorosea
. Spore con-
centrations are not equal due to the differ-
ent developmental rate of each fungus (Kon-
todimas and Gkotsi, 2009). Spore viability
measured over 96% for all selected strains.
Insects
Both adult and larvae of the sisal wee-
vil were collected from ornamental aga-
