Spiroplasmas as Biological Control Agents of Insect Pests
Toward development of spiroplasmas as novel toxin-delivery systems for biocontrol of beetle pests in the United States (Leptinotarsa decemlineata) and Israel (Maladera matrida), media for cultivating beetle-associated spiroplasmas were improved and surveys of these spiroplasmas were conducted to provide transformable strains. Extensive surveys of spiroplasmas yielded promising extrachromosomal elements for vector constructs. One, plasmid pCT-1, was cloned, characterized, and used as a source of spiroplasma origin of replication in our shuttle vectors. The fibrillin gene was isolated and sequenced and its strong promoter was also used in the constructs. Means for transforming these vectors into spiroplasmas were developed and optimized, with electroporation found to be suitable for most applications. Development and optimization of means for using large unilamellar vesicles (LUVs) in spiroplasma transformation represents a breakthrough that should facilitate insertion of large clusters of virulence genes. With completion of the vector, we should thus be poised to genetically engineer spiroplasmas with genes that will express toxins lethal to our target beetles, thus providing an effective and inexpensive alternative to conventional means of beetle control.