Basic HTML Version
Bt Research 2013, Vol.4, No.1, 1
-
7
ISSN 1925-1939
http://bt.sophiapublisher.com
4
It’s important to comment that Cry1 and Cry2 toxins
are the most studied toxins and that the
cry1
gene is the
most abundant in the nature (Bravo et
al., 1998,
Rosa-Garcia et
al., 2008).
In the cause of S2122 strain, it didn’t present the 65
kDa protein because it probably could have silent
genes and this characteristic will be studied with more
details in the future. The high toxicity of the strains to
P. xylostella
suggests that the Cry1Ab
,
Cry1Ac and
Cry
1C
toxins present in the strains can be responsible
to toxicity against this insect (Monnerat et al., 1999).
This crystals´ morphology of the strains was similar to
B. thuringiensis kurstaki
HD-1 as described
previously (Praça et al., 2004, Monnerat et al., 2007,
Santos et al., 2009, Sezen et al., 2010). The
bypiramidal crystals may be related to the presence of
Cry1 proteins and show the activity against
lepidopteran and coleopteran insects, the cuboidal are
associated to Cry2 proteins, which show the activity to
lepidopterans and dipterans (Dankocsik et al., 1990,
Wu et al., 1991, Bravo et al., 1998, Praça et al., 2004,
Monnerat et al
.
, 2007, Xie et al., 2010). Cuboidal
crystals appear in the studied strains, although this
crystal is known as toxicity to coleopteran insects
B.
thuringiensis
strains characterized in this study can be
used in the formulation of new bioinsecticide to
manage the insect’s resistance and it’s also possible to
be used endophytically for controlling
P. xylostella
.
3 Materials and Methods
3.1 Strains and culture conditions
Three strains of
B. thuringiensis
from the Collection
of Invertebrate Bacteria of Embrapa Genetic
Resources and Biotechnology were used in this work.
These strains were obtained from soil and water
samples from different parts of Brazil and were
previously identified as pathogenic to Lepidopteran
species of insects (http://plataformarg.cenargen.
embrapa.br/rede-microbiana/colecoes-de-ulturas/colec
ao-e-bacterias-de-invertebrados/colecao-de-bacterias-
de-invertebrados).
B. thuringiensis
subspecie
kurstaki
(Btk) HD-1, obtained from the Collection of
Bacillus
thuringiensis
and
Bacillus sphaericus
of the Institute
Pasteur, Paris, was used as standard.
All strains and HD-1 were grown in Embrapa medium
(Monnerat et al., 2007), for 72 h., 200 rpm, 30
℃
Then, they were centrifuged at 12,800
x
g for 30 min,
at 4
℃
(BR4i centrifuge Jouan). The pellets,
containing spores and crystals were frozen for 16 h
and lyophilized for 18 h in Labconco model
Lyphlock18 freeze-dryer.
3.2 Bioassays against
P. xylostella
Bioassays were carried out using second instar larvae
of
P. xylostella
reared in the laboratory at (26 ± 2)
℃
,
(60 ± 10)% humidity and photophase of 12 h
(Medeiros et al., 2003). Two kinds of bioassays were
carried out, the selective one to confirm the toxicity of
the strains and the quantitative one to determine the
lethal concentration required to kill 50% of the tested
insects (LC
50
).
The selective bioassays were performed by the
procedure described by Tabashnik
et al
(1990
)
with
some modifications. Pieces of cabbage (
Brassica
oleracea
L.) leaves (6 cm × 3 cm) were dipped
vertically for 10 minutes in spore/crystal powder
diluted 10% in distilled water. Leaves were then air
dried vertically for 1 h at 25
℃
then placed in a Petri
dish (80 mm× 15 mm) with 10 larvae, in a total of tree
replicates. The procedure of the selective bioassay was
the same for each strain. After 48 h the surviving larvae
were transferred to a new leaf and the mortality was
assessed. Larval mortality was assessed again at day
five (Monnerat et al., 1999) and the rate of mortality
was determined and compared by variance analyses
using Sigmastat analyses statistic (Kuo et al, 1992).
In order to determine the LC
50
, 6 concentrations
(between 100 µg/mL and 0.01 µg/mL) of the
lyophilized mixture of spores and crystals were
diluted in sterile water. Then, the cabbage leaves were
dipped in each concentration as described for the
previous bioassay. The bioassays were repeated three
times. The mortality data were evaluated on the fifth
day analyzed by Probit analyses and LC
50
(Robertson
et al., 2002) was determined and analyzed by variance
analyses (Kruskal-Wallis) using Sigma Stat analyses
statistic (Kuo et al., 1992).
3.3 Characterization of the
B. thuringiensis
strains
toxic to
P. xylostella
3.3.1 SDS-PAGE (Polyacrylamide gel electrophoresis)
The proteins were extracted according to Lecadet et al