Basic HTML Version
Bt Research 2013, Vol.4 No.2 8-13
ISSN 1925-1939
http://bt.sophiapublisher.com
9
Despite the good prospects for the control of blackfly,
there are few commercial products and the high cost
of importation may limit their use in biological control
programs. To facilitate local production of a
bioinsecticide for production and use against
Simulium
in Brazil, this study investigated the activity
of standard strains and new isolates of
Bt
from Brazil
to identify the most effective strains for control.
Accordingly, the present study aimed to isolate and
characterize
B. thuringiensis
strains toxic to
Simulium
spp. from the blackfly-endemic Amazon
region, which is known for its high biodiversity, and
to compare their toxicity with strains from serotypes
israelensis
,
medellin
,
jegathesan
, known to be toxic
to dipteran larvae.
1 Results and Discussion
From the 200 soil samples tested, 96 strains of
B.
thuringiensis
were isolated. These strains were named
and deposited in the Collection of Bacteria of
Invertebrates belonging to EMBRAPA Genetic
Resources and Biotechnology. Due to the limitations
of culturing
Simulium
on a large scale, selective
assays to identify dipteran-active strains from the
collection were carried out using the larvae of the
mosquitoes
Aedes aegypti
and
Culex quinque-
fasciatus
. Among the 96 strains from Amazonia, only
two, S2271 and S2272 caused 100% mortality in
selective bioassays and were selected for use in
further assays against blackflies. The other 94 caused
less than 50% mortality.
In taxonomic analysis of the larvae of
Simulium
spp.
collected from the field for bioassay, over 90% of the
specimens were identified as
S. perflavum
Roubaud.
Ten
B. thuringiensis
strains were tested against
Simulium
: the two dipteran active Amazonia strains
above; a number of other Brazilian strains from the
EMBRAPA collection representing different
Bt
serotypes; and three standard strains. Of these 10
strains, 6 caused 100% mortality of larvae of
S.
perflavum
in selective bioassays (S2271 and S2272
from Amazon and S1785 (from EMBRAPA) and
Bt
medellin
163-0131 (from Colombia), and
Bt
jegathesan
T28 A001 and
Bt
israelensis
T14001 (from
Institute Pasteur). Strains belonging to serotypes
aizawai
(S1576),
kurstaki
(S1905 and S1450) and
tenebrionis
(S1122) showed no activity against the
blackfly larvae. The 6 toxic strains were then
subjected to further bioassay, to determine the lethal
concentration that kills 50% of the target population
(LC
50
). Strains S1785, S2271 and
Bti
were the most
toxic, presenting LC
50
values between 9.5 mg/mL and
15.1 mg/mL. The strain S2272 showed an LC
50
of
18.3 mg/mL, statistically different from the first group,
and
Bt
jegathesan
and
Bt medellin
were grouped in a
third group of toxicity (lower) showing LC
50
values of
89.1 mg/mL and 108.2 mg/mL, that were statistically
indistinguishable, but low compared with the most
toxic group (Table 1).
Table 1 Characterization of
Bt
strains toxic to
Simulium
spp.
larvae: Serotype and LC
50
(mg/mL)
Strains
Serotype
LC
50
(mg/mL)
S2271
israelensis
9.5 (7.1~12.4)
S1785
israelensis
10.5 (7.2~12.6)
Bti
T14001
israelensis
15.1 (10.3~21.8)
S2272
auto-agglutinant
18.3 (13.1~26.4)
Bt
163-0131
medellin
89.1 (72.2~106.3)
Bt
T28 A001
jegathesan
108.2 (81.2~136.5)
The results of serological characterization showed that
one of the Amazon strains belongs to serovar
israelensis
(S2271) and the other (S2272) is
auto-agglutinating (Table 1). Strains S1785, S2271
and S2272 showed a protein profile similar to that of
the standard
Bti
T14001 strain presenting proteins of
molecular weight around 130 kDa, 72 kDa and 30 kDa,
consistent with
Bti
-like proteins Cry4A and Cry4B
(130 kDa), Cry10 and Cry11 (72 kDa) and Cyt1 and
Cyt2 (30 kDa) (Figure 1). All these proteins are
effective against dipteran larvae (Monnerat and Praça
2006). The protein profiles of the strains belonging to
serotype
medellin
and
jegathesan
were distinct from
those of serovar
israelensis
.
The two Amazon strains (S2271 and S2272) presented
PCR products of the expected sizes when amplified
with probes for
cry4A
,
cry4B
,
cry11
,
cyt1
and
cyt2
genes. These results complement the protein profile
obtained for the strains showing similarity between
these strains and serovar
israelensis
.