Construction of a potent strain of Bacillus thuringiensis against the cotton leaf worm Spodoptera littoralis

This work has been carried out in order to construct a potent Bacillus thuringiensis (Bt) strain active against the Egyptian cotton leaf worm S. littoralis. Toward this target, the d-endotoxin crystal protein genes cry1C (that encodes an insecticidal protein highly specific to S. littoralis) and cry1Ag (1Ac like) were used. HindIII digested cry1C was ligated into the HindIII site of the shuttle vector pHT7593 yielding the plasmid pHTNC3. HincII digested cry1Ag was ligated into SmaI site of pHT7593 and into SmaI site of pHTNC3. The three plasmid constructs were used to transform E. coli strain JM109. Colonies likely to contain these recombinant plasmids were screened for the production of toxin proteins. Positively identified transformants produced the expected size protein, detected by partial purification of protein, and is truncated upon trypsin digestion. The pHT7593-bearing cry1C, pHT7593-bearing cry1Ag and pHTNC3-harboring-cry1Ag were transferred into the non-crystalliferous (Cry-) Bt4 Bt strain. The introduction of the cry1Ag gene into Cry- Bt4 resulted in the formation of bipyramidal crystals. The introduction of both cry genes 1C and 1Ag resulted in the multiplication of bipyramidal crystals. In bioassays, cry1Ag-expressing BT4 Bt strain caused mortality of S. littoralis larvae only slightly (the LC50 was 104 ppm). In the presence of only Cry1C, the LC50 was 64 ppm. In presence of Cry1C co-expressed with Cry1Ag the LC50 decreased to 2.2 ppm. Thus, a combination of the Cry proteins 1C and 1Ag could result in effective insect control. With this approach, a combination of Cry proteins can be designed rather than discovered.