Paratransgenesis: The dynamics of engineered Enterobacter symbionts and Cry1Ac-producing Enterobacter for biocontrol of Helicoverpa insect pests in crop production

The potential application of insect gut microorganisms in paratransgenic crop pest management techniques is being investigated. The Helicoverpa gut is known to harbor the symbiont Enterobacter. However, little is known about the dynamics of Enterobacter in the Helicoverpa gut, its interaction with the phyllosphere, and the mechanisms by which it is delivered to this Lepidopteran species. To evaluate the insecticidal activity of Cry1Ac and its capacity to colonize the Helicoverpa gut and the tomato phyllosphere, the symbiont Enterobacter, which was isolated from the Helicoverpa gut, was transformed with constructs 2 (cry1Ac-KmR-pUC18) and 3 (KmR-pGFP). Within 48 hours and 24 hours, respectively, the Helicoverpa gut and tomato phyllosphere were effectively colonized by the GFP-producing construct 3-Enterobacter. In insect bioassays, Cry1Ac-producing constructs 2-E. coli (K12) and 2-Enterobacter were used for diet and leaf-dip testing. Helicoverpa neonates died 86-100% when fed diets supplemented with 10 -10 cells of Cry1Ac-producing construct 2-E. coli (K12), whereas 79-100% died when fed diets supplemented with 10 -10 cells of Cry1Ac-producing construct 2-Enterobacter. Furthermore, when the Cry1Ac-producing construct 2-Enterobacter (0.10 x 109 cells) was applied to the tomato phyllosphere, it caused 100% mortality of neonates within 192 hours. The successful vector modification, genetic transformation, and establishment of recombinant Enterobacter cells in the guts of Helicoverpa larvae and on tomato leaves lay the foundation for advancing a paratransgenic strategy. This engineered bacterium could potentially replace synthetic insecticides for managing lepidopteran pests in crops.