EXPRESSION OF A BACILLUS-THURINGIENSIS DELTA-ENDOTOXIN GENE BY BACILLUS-PUMILUS

The delta-endotoxin genes from Bacillus thuringiensis were introduced into a rhizosphere-inhabiting Bacillus pumilus isolate to create a del ta-endotoxin expression and delivery system for subterranean feeding i nsects such as the larvae of pale western cutworm (Agrotis orthogonia Morrison (Lepidoptera: Noctuidae)). Preliminary experiments indicated that Bacillus thuringiensis subsp. kurstaki cultures were toxic to pal e western cutworm larvae. Three different cry genes from Bacillus thur ingiensis subsp. kurstaki were cloned into high and low copy number ve ctors and mated into Bacillus pumilus RB8. When carried on high copy n umber vectors, cry genes appeared to inhibit sporulation and delta-end otoxin production in Bacillus pumilus RB8 cultures, since microscopic examination of these cultures revealed that <0.1% of the cells of late stationary phase cultures had sporulated and produced parasporal incl usions. On low copy number vectors, the cry genes did not inhibit spor ulation; however, production of delta-endotoxins was undetectable. Usi ng a heat shock regime for enrichment of sporogenous crystalliferous v ariants, a Bacillus pumilus isolate, carrying cryIA(c) on a high copy number plasmid was obtained in which high level delta-endotoxin produc tion occurred concomitant with sporulation. Synthesis of functional de lta-endotoxin by this strain was confirmed by Western blot analysis an d bioassay with pale western cutworm larvae. These results show that r hizosphere-inhabiting bacilli are indeed a potential route for introdu ction of delta-endotoxins to the root environment for biocontrol purpo ses.