الفهرس 
Biological control approachOnly 14 pages are availabe for public view
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Abstract
Bacillus thuringiensis (Bt) is a spore-forming bacterium wellknown
for its insecticidal properties associated with its ability to produce
crystal inclusions during sporulation. These inclusions are proteins
encoded by cry genes and have shown to be toxic to a variety of insects
and other organisms like nematodes and protozoa.
Lepidoptera causes some of the most devastating insect pests for
important crops in Egypt; their control is a priority as well as a necessity.
In this context, use of environmentally safe technology to reduce crop
damage like B. thuringiensis would be extremely valuable. Consequently,
this study was planned to establish and characterize a collection of Bt
isolates from samples collected in different Egyptian localities in order to
find novel strains toxic against insect pests of economically important
crops.
The results of this study were as follows:
1. Bacterial samples were collected from soil of sixteen Egyptian
governorates(from January 2013 to May 2014).
2. Heat treatment of soil samples and aerobic incubation were
followed to select spore-forming bacteria from global bacterial
flora.
3. The acetate selection protocol was used to isolate B.
thuringiensis from a mixed population of soil bacteria
depending on the fact that germination of B. thuringiensis spores
was selectively inhibited by sodium acetate. After treatment, all
of the non-sporulated microbes were eliminated by heat
treatment at 80oC.4. Bacillus thuringiensis was selected according to colony
morphology (round, about 3-5 mm in diameter, raised or flat
slightly shiny, grayish white opaque with entire edge and having
a granular surface.
5. After purification of the bacterial isolates, the microscopic
examination showed that all the 16 Bacillus isolates are Grampositive
rod shaped bacteria.
6. The amount of acetone powder (containing spore-crystal
mixtures)produced from each isolate differed considerably and
ranged from 58 mg to 288.5 mg. This variation could be
attributed to the variation in doubling time of these different
Bacillus strains and/or the differences in their rate of
sporulation.
7. The highest three amounts of acetone powder were 288.5,260.6
and 249.8 which were obtained from isolates number 3, 14 and
13, respectively. On the other hand, the lowest three amounts of
acetone powder were 58, 60.5 and 70.5, which were obtained
from isolates number 2, 7 and 4, respectively.
8. SDS-PAGE analysis showed that bands were detected with
molecular weight at 130, 65 and 35 KDa. The band with 130-140 KDa is present in almost all the tested Bacillus strains. On
the other hand, bands with molecular weights of 65 and 35 KDa
are observed in only few of the tested strains.
9. All of the 16 Bacillus strains exert a percentage of mortality
comparing to the control (0 %),nevertheless, the degree of
toxicity varied greatly among them (13.3 to 100 % mortality).
10. The lowest toxic strains were number 14, 11 and 6 which
resulted in mortality percent of 13.3, 16.6 and 23.3 %,respectively. Meanwhile, relatively high mortality percentage
(66.6 %) was obtained using the crystal proteins of Bacillus
strains number 2, 3, 10 and 13. On the other hand, strains
number 9 and 15 showed appreciable mortality of 100 % and
96.6 %, respectively.
11. Strains number 9 from Damietta and number 15 from Sharkia,
which caused the highest mortality of cotton leaf worm larvae,
were retested for their insecticidal activity towards the 1st
instars larvae of potato tubers moth (Phthorimaea
operculella). These two strains showed mortality of 7 % and 3
%, respectively.
12. The total DNA prepared from the selected isolates (9 and 15)
was subjected to PCR analysis to identify cry I, cry III and cry
IV genes. Strain number 9 amplified a single fragment of
about 490 bp indicating that this strain would belong to cry I
gene family. Results also reveal that strain number 15 from
Sharkia did not yield any product with the three primers used
in this study.
13. Strains number 9 and 15 of B. thuringiensis were investigated
by electron microscope for crystal protein morphology. Strain number 9produced bipyramidal crystal proteins, consistent
with the presence of Cry1 protein. On the other hand, strain
number 14 from sharkia produced spherical shaped crystals.
14. PCR product of 16S rRNA gene from strains number 9 and 15
which showed potentially toxicity to cotton leaf worm
(spodoptera litoralis) was sent to Sigma Company for
sequencing. The partial 16S rRNA sequences were tested
using National Center for Biotechnology Information (NCBI)
database.15. Results of sequence alignment of B. thuringiensis strain
9showed 99% of identity with the sequence from Bacillus
thuringiensis Bt 407.Strain 9 also shared a close relationship
with others Bt strains on the database.
16. Results of sequence alignment of B. thuringiensis strain15
showed 98% of identity with the sequence from Bacillus
thuringiensis ATCC 10792 strain. This strain also shared a
close relationship with other Bt strains on the database.
The discovery of a highly toxic isolates reveals the usefulness of
screening studies for novel Bt strains. The future application of these
strains in biological control programs requires optimization of the
production conditions of the microorganisms using low-cost substrates. In
this context, characterization of phenotypic and biochemical and
molecular properties as evaluated in this study is highly relevant.