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Bt Research 2015, Vol.6 No.1 1-8
ISSN 1925-1939
http://bt.biopublisher.ca
3
cry1Fa
+
vip3Aa
; 33 (28.95%) amplified the
combination of
cry1Fa
+
chi
; and 1 (0.88%) amplified
the
vip3Aa
+
chi
combination (Figure 2). The PCR
detection of the
cry
,
vip
, and
chi
genes revealed that
cry1Fa
and
chi
had the highest gene profile
frequencies in the collection.
Figure 2. The frequencies of the
cry1Fa, vip3Aa
and
chi
genes
(Lepidoptera-specific) in 114 isolates of
Bacillus thuringiensis
.
1.2 Polymorphism analysis of the
cry1Fa
,
vip3Aa
,
and
chi
genes via PCR-RFLP
After the
cry1Fa
,
vip3Aa
, and
chi
genes were
amplified from the isolates, we used the PCR-RFLP
technique to analyze the polymorphisms in the
sequences from the 12 isolates from which all three
genes had been successfully amplified.
No differences in the obtained banding patterns were
found among any of the isolates. This result suggests
that there are no polymorphisms in the amplified
fragments that could be detected using the applied
restriction enzymes; therefore, it was not possible to
determine any associations between polymorphisms
and the mean mortality of
S. frugiperda
.
1.3 Bioassays
None of the
B. thuringiensis
isolates used in the
bioassays caused 100% mortality among the
S.
frugiperda
larvae, but several isolates exhibited
efficacies greater than 70%, which can be considered
efficacious (Campanini et al., 2008). The mortality
rates varied among the different gene combinations
throughout the evaluation period (Table 2). This
finding suggests that the effectiveness in causing
mortality may be related to a synergistic mechanism
among the three genes, which was also found by
Costa et al. (2010) for
Aedes aegypti
(L.) (Diptera:
Culicidae).
All the treatments that contained the
cry1Fa
+
vip3Aa
+
chi
gene combination produced mortality rates
between 60% and 94%. Isolates I_10 and I_17 caused
the highest mortality (Table 2). Additionally, there
were differences in mortality between the treatments
with the pair-wise combinations
cry1Fa
+
vip3Aa
,
cry1Fa
+
chi
and
vip3Aa
+
chi
; these combinations
resulted in 80%, 12%, and 62% mortality, respectively.
In comparison, when applied individually,
cry1Fa
,
vip3Aa
, and
chi
caused 16%, 29%, and 2% mortality,
respectively, which was significantly different
compared with the isolates containing two or three
genes (Table 2). Thus, it was found that
vip3Aa
,
cry1Fa
, and
chi
alone were not effective in controlling
S. frugiperda
, which was also true for the
cry1Fa
+
chi
combination. Furthermore, the synergy of the
chi
+
vip3Aa
combination appeared to produce a stronger
effect compared with the treatment with
vip3Aa
alone.
Neither the Coleoptera-specific standard strain
B.
thuringiensis
var.
tenebrionis
, which was used as a
negative control, nor the blank (water) treatment
caused any larval mortality (Table 2).
2 Discussions
The research on
B. thuringiensis
conducted during the
past 20 years has shown that this species is more
complex than was originally thought. Rather than
killing insect larvae only with the well-known
insecticide crystal proteins (ICP), this bacterium also
produces a series of other substances that aid in
increasing mortality, including
-exotoxin, chitinase,
hemolysin, phospholipase C, vegetative insecticidal
proteins, and most likely other compounds that are yet
to be discovered (Schnepf et al., 1998; Donovan et al.,
2001; Lin and Xiong, 2004 ).
The pioneering research that characterized the chitinase
protein distribution in
B. thuringiensis
was performed
by Liu et al. (2002), who showed that chitinase was
produced by
B. thuringiensis
isolates and that some of