Microcosm method to assess survival of recombinant bacteria associated with plants and herbivorous insects

A microcosm method was developed to investigate survial and fate of genetically engineered bacteria associated with plant surfaces and a plant-feeding insect, the variegated cutworm,Peridroma saucia. Larvae on radish plants in microcosms were sprayed with nonrecombinantPseudomonas cepacia and a recombinant strain ofP. cepacia carrying the transmissible plasmid R388::Tn1721. Leaf, whole insect, foregut, and frass samples were periodically assayed over a 48-h period to enumerate total bacteria andP. cepacia strains. Immediately after spraying,P. cepacia comprised about 20%–30% of the total population on leaves, which was 2×10⁷ cfu/g of leaf. Approximately 4×10⁷ total cfu were recovered from each gram of whole insect, when theP. cepacia strains averaged about 3×10⁵ cfu/g. After 2 days, the total epiphytic population had increased approximately fourfold, while theP. cepacia strains had decreased to 2%–30% of their initial numbers. After 24 and 48 h, eachP. cepacia strain numbered between 10⁴ and 10⁵ cfu/g of insect in foregut samples, whereas none was detectable in frass. Plasmid transfer fromP. cepacia R388::Tn1721 to the nonrecombinant recipientP. cepacia strain was not observed.