Bt Research 2012, Vol.3, No.3, 11
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19
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be observed for strains S1905, S2122 and S2124 as
well as for the standard strain S1450. Structures from
all the
B. thuringiensis
strains tested were visible
adhering to the surface of leaves and in their
depressions, being concentrated around the stomata.
They were also seen in the stomata chamber. The
colonization on stomach and nearby areas of cabbage
leaves were observed in this work and similar results
were found by Silva-Neto et al (2006) in the
penetration of
Acidovorax avenae
subspecies
citrulli
by stomata openings in leaves of yellow melon
(
Cucumis melo
).
The bacterial cells were found in areas protected by
cuticular flanges or depressions and around the
stomata; these events have already been observed for
other microorganisms, such as phyto-bacterioses and
nitrogen fixers (Silva-Neto et al., 2006; Mattos et al.,
2008).
The formation of fibrils was also observed by
Silva-Neto et al (2006) and Campbell et al (1987) that
observed the presence of fibrilar material, favoring
adherence to the surface of hosts such as yellow
melon (
Cucumis melo
)
and canola (
Brassica napus
L.
var
.
oleífera
).
The adherence process is not completely
understood, but it is considered essential in the
infection process, because it helps bacteria to stick to
host surfaces, aiding the process of penetration and
colonization.
All the strains of
B. thuringiensis
that were used in
this study were successful in colonizing cabbage leaf
tissues, but structures of strains S1450 and S1905 of
B.
thuringiensis
were seen in more abundance. The latter
is a Brazilian strain known to be toxic to larvae of
insects from the Lepidoptera order (Monnerat et al.,
2006).
These strains were visible in the stomata
openings, producing cells, spores and crystals, thus
demonstrating germination, sporulation and formation
of crystals of
B. thuringiensis
.
The fact that
B.
thuringiensis
was observed in all parts of the seedlings
was of great importance for this study, because it
shows the possibility of using
B. thuringiensis
in the
control of agricultural pests that cause leaf fall,
endophagous insects, sucking insects, soil pests and
phytopathogenic vectors.
It is likely, then, that strains S1450 and S1905 are
good colonizers of cabbage seedlings, standing out for
their intense colonization in all parts of the seedlings,
as well as for the presence of bipyramidal, cuboid and
spherical crystals, which are essential in toxicity to
insect pests. Thirty days after being immersed,
bacterial structures such as vegetative cells, spores and
crystals of
B. thuringiensis
were found in all parts of
the analyzed seedlings, proving that
B. thuringiensis
penetrates and colonizes tissues of cabbage seedlings.
The confirmation of effective colonization of seedlings
by
B. thuringiensis
demonstrates that this bacterium
may be used endophytically to control
P. xylostella
,
a
cryptic pest that causes damage to brassica crops in
Brazil and worldwide.
Scanning electron microscopy produced proof that
B.
thuringiensis
had penetrated the roots, stems and
leaves of cabbage seedlings. It was confirmed that the
methodology for inoculating
B. thuringiensis
in seeds
was appropriate, providing conditions for observing
the structures of this bacterium in the cabbage
seedling tissues. This is the first demonstration of
colonization of cabbage seedling tissues by Brazilian
strains of
B. thuringiensis
using scanning electron
microscopy.
The strains of
B. thuringiensis
did not promote growth
in the cabbage seedlings, but they did present
endophytic behavior without inducing disease symptoms.
The
B. thuringiensis
strains marked with methionine
35
S were detected in all parts of cabbage seedlings.
Similar results were found by Monnerat et al (2009) in
kale and cotton plants inoculated with
B. thuringiensis
HD-1
labeled by methionine
35
S.
This colonization in cabbage seedlings is of great
importance, reinforcing as it does the possibility of
using
B. thuringiensis
endophytically in the control
of
P. xylostella
,
a pest which, when the cabbage head
forms, enters it and causes damage to the plant,
reducing its value and lowering productivity. This
corroborates the results of Monnerat et al (2009), in a
study which demonstrated the presence of
B.
thuringiensis
in cotton plant tissues for several
weeks by means of cell counts after treating them
with Btk HD
-
1.
The seed is one of the main vehicles for spreading
microorganisms, and therefore disseminating
B.
thuringiensis
via seeds should be more thoroughly
investigated, so that this inoculation method can be
