3.

Results and discussion

3.1 DNA patterns of B. thuringiensis strains
In DNA patterns of B. thuringiensis strains, we can difference them by observe the plasmid content and for
novel strain we can discover the novel cry gene content. To get the strain with novel properties of 200 B.
thuringiensis, we can isolate them with Tunisian soil. After DNA extraction and electrophoresis on agarous
gel, we will obtain polymorphism among the DNA profiles, except for BLB459. BLB459 is unique, it is
deeply investigated. PFGE test used to compare the DNA smalmacrorestriction profile of B. thuringiensis
BLB459 with BUPM95 and HD1. The strain of BLB459 has more specific plasmid profile rather than the
strain on HD1. To know the phylogenetic relationships of BLB459 with HD1 and BUPM95, we can draw
a dendrogram based on their numbers and sizes bands from small-macrorestriction PFGE test. 100% of
similarity was observed between the HD1 and BUPM95, because both of them belong to subspecies of B.
thuringiensis. But, the BLB459 only has 46,16% of similarity with these reference strains. So, we can
conclude that Smal-macrorestriction is very effective to differentiate the B. thuringiensis strains.
3.2 Crystal Protein Content
SDS-PAGE analysis is used to compare the composition of parasporal proteins of B. thuringiensis type
BLB459, BUPM95, and HD1. BLB459 contain three major cry proteins, about 140-150 kDa and 70-75
kDa molecular weight. BUPM95 and HD1 produced 2 major cry proteins with 130-135 kDA and 65-70
kDa molecular weight. According of the analysis, BLB459 strains was able to produce different cry-type
proteins, other than BUPM95 and HD1.
3.3 Cry Gene content
Due to the unusual crystal protein profile of BLB459, so we investigate the cey gene content. PCR
amplificarion test showed that BLB459 dont has cry1A, cry2A, cry3A, and cry II. We found that BLB459
lacking the 130-135 kDa and 65-70 kDa were corresponding with cry 1 type and cry 2 type. By PCR
amplification assay, we found that BLB459 has cry 30, cry 40, and cry 54 type genes and giving PCR
fragment with molecular weights of 1463,886 and 1476 bp. After the PCR fragments wre sequenced and
analyzed by blast nucleotide program, we get data that the nucleotide sequences of the cry genes generated
by promer pairs Up30-5/Up30-3 and Up39.405/Up39.40-3 harbor high homologies with cry30E (91%) and
cry40A (95%).By using S54-5 and S54-3 primers to generate the partial nucleotide sequence of the
fragment, we will have cry 54Ba gene. PCR-RFLP is important to identify the existence of known cry genes
and novel cry genes of B. thuringiensis strains.
3.4 Insecticidal activity of BLB459 againts Lepidoptera
The parasporal proteins of BLB459 and HD1 exhibited high toxicities to E. kuchniella and S. littoralis
larvae. However, the novel selected strain (BLB459) has higher potential against the polyphageous
Lepidoptera S. littoralis with an LC50 of 80(69-91) g/cm2. So, we can use BLB459-endotoxins to control S.
littoralis.
3.5 Histopathological Effects of Cry toxins in Lepidoptera
The histopathological effects of BLB459 cry toxins on Lepidoptera were studied because of their toxicity.
In untreated E. kuehniella and S. liitoralis larvae, their midgut have uniform morphology and well-defined
epithelial cells. But when the larvae treated with BLB459 cry toxins, the midgut ephitelium get extensive
damages such as vacuolization of the cytoplasm, brush border membrane lysis, vesicles formation, and
disintegration of the apical membrane. It means that the E. kuehniella and S. littoralis midget tissue is
disintegrated by the B. thuringiensis cry toxins and cause the larvae die. BLB459 found to be an original
strain because it has typical plasmid profile and cry genes content. B. thuringiensis gives an important
histopathological effects against Lepidoptera larvae, so we can use BLB459 to become the new
bioinsectides.