Diversity of Bacillus thuringiensis isolates from Egyptian soils as shown by molecular characterization.

Different techniques were adopted for molecular characterization of several indigenous strains of Bacillus thuringiensis (Bt) previously isolated from Egyptian soil samples. These isolates show different toxicity levels against neonate larvae of both insect species; Spodoptera littoralis (Biosduval); and Helicoverpa armigera (Hübner). The parasporal crystals among the most potent isolates contained polypeptides of about 127 and 130 kDa. PCR screening for genes encoding different Cry genes was performed. The Cry 1 gene is the most abundant in these isolates (83.33%) among tested Cry-type genes, followed by Cry 1 gene subfamilies (Cry 1B and Cry 1C) with percentage of 38.88% and 77.77%, respectively. The tested isolates showed the presence of Cry 2A(a,b) gene, but not all of these isolates were positive for Cry 2 gene (55.55%). Only 27.77% and 16.66% of the tested isolates harbor Cry 4 and Cry 3 genes, respectively. All strains were negative in PCR assays for the Vip 3Aa1 gene. Moreover, DNA fingerprinting using RAPD-PCR was performed to detect the genetic similarities and dissimilarities among the different isolates and standard strains. Assessment of Bt diversity based on the combined analysis of their protein and RAPD-PCR banding patterns was performed. This study demonstrates that Bt strains isolated from Egyptian soil samples can be distinguished and identified on the basis of the distribution of Cry-type genes and RAPD fingerprints.

Histopathology of the larval midgut of Helicoverpa armigera (Hübner) fed on Bacillus thuringiensis crystals and Bt-tomato plants.

The histopathological effects of the spore-crystal complex of indigenous Bacillus thuringiensis (Bt) isolate, as well as Cry 2Ab gene expressed in transgenic tomato plants on the midgut of 4th instar larva of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidea) has been investigated using the transmission electron microscope (TEM). Remarkable ultrastructural changes were observed in the columnar and goblet cells of the larval midgut after feeding on either transgenic tomato leaves, or spore-crystal complex of Bt. The effects observed included breakdown of microvilli of epithelial cells, increase in the electron density of the cytoplasm and vacuolation associated with different sizes of lysosomes; interruption of the goblet cells and distorted goblet cavities which lost their cytoplasmic projections; destruction of the mitochondria which lost their cristae; degeneration of the endoplasmic reticulum; collapse of the nucleus associated with rupture of nuclear envelope and clumped chromatin. Feeding the larvae on transgenic Bt-tomato plants caused in addition to the aforementioned changes severe vacuolation and degeneration of the nucleus in both columnar and goblet cells and the nuclear membrane was broken into electron dense ring spheres.