Basic HTML Version
Bt Research 2013, Vol.4, No.1, 1
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7
ISSN 1925-1939
http://bt.sophiapublisher.com
2
1 Results and Analysis
1.1 Bioassay of the
B. thuringiensis
strains against
P.
xylostella
The three strains S1905, S2122 and S2124 were toxic
to
P. xylostella
. S1905, S2122 and HD-1 caused 100%
of mortality after 48 hs, although S2124 caused
58.33% of mortality after 48 hours (Kruskal-Wallis:
H
3
=10.8, P =0.013) and 98.33% of mortality after five
days (Kruskal-Wallis: H
3
= 3.0, P = 0.392) (Table 1).
Table 1 Perceptual of mortality of second instar larvae of
P.
xylostella
(media ± standard deviation) caused by Brazilian
B.
thuringiensis
strains
Strains
Percentual of mortality (%)
48 hours
96 hours
S1905
100 ± 0a
100 ± 0a
S2122
100 ± 0a
100 ± 0a
S2124
58.33 ± 2.877b
98.33 ± 2.877a
S1450-Btk padrão
100 ± 0a
100 ± 0a
Note: Measures followed by the same letter, in the columns, do
not differ among themselves
After selective bioassays, the insects were submitted
to lethal concentration bioassays. The CL
50
of the tree
B. thuringiensis
strains varied between 2.336 to 4.842
µg/ml (Table 2) and all of them were similar to HD-1
(ANOVA: F = 0,673, P = 0,595).
Table 2 Estimation of Lethal Concentration (LC
50
) of
B.
thuringiensis
strains toxic to second instar larvae of
P.
xylostella
after five days
Strains
CL
50
(ng/mL)
Fiducial limit (95%)
S1450 HD-1
4.202±0.558a
0.556 – 5.600
S1905
3.430±1.513a
1.381 – 5.574
S2122
2.326±1.732a
0.421 – 24.176
S2124
4.842±3.261a
1.932 – 13.4987
Note: Measures followed by the same letter, in the columns, do
not differ among themselves by the fiducial limit
1.2 Characterization of
B. thuringiensis
strains
toxic to
P. xylostella
The analysis of the spore-crystals mixtures by
SDS-PAGE showed the presence of two proteins of
130 and 65 kDa in S1905 and S2124 strains and in
S2122 showed only the 65 kDa protein (Figure 1).
S1905 produced amplicons for genes
cry1Aa, cry1Ab,
cry1Ac, cry1B, cry2Aa
and
cry2Ab
(Table 3). S2122
produced amplicons for seven genes
cry1Aa, cry1Ab,
cry1Ad, cry1C, cry1D, cry1F
and
cry
2
Ab.
S2124
strain produced amplicons for
cry1Ab, cry1E, cry2Aa
and
cry2Ab
(Table 3).
Figure 1 Protein profile of
B. thuringiensis
strains HD1
,
S1905,
S2193 e S2124
Note: 1: Full Range Rainbow molecular weight marker (GE), 2:
HD-1, 3: S1905, 4: S2122 e 5: S2124
1.3 Scanning and Transmission electron microscopy
of
B. thuringiensis
strains
The morphological characterization through scanning
and transmission microscopy showed three different
types of crystal protein inclusions. The strains
produced bipyramidal, cuboidal and spherical crystals
(Figure 2; Figure 3).
Figure 2 Scanning Electronic Microscopy of spore-crystals of
Bacillus thuringiensis
strains
Note: A - S1905, B - S2122, C - S2124 and D - S1450
B.
thuringiensis
subsp
. kurstaki
HD-1, ce - spherical crystal, cb –
bipyramidal crystal, cc - cuboidal crystal, ep - spore
2 Discussion
The three strains S1905, S2122 and S2124 were tested
against
P. xylostella
in selective bioassay showing
toxicity.
In the first 48 hours, S1905 and S2122 strains
killed the insects faster than S2124 when compared