Introduction of a Lepidopteran-Specific Insecticidal Crystal Protein Gene of Bacillus thuringiensis subsp. kurstaki by Conjugal Transfer into a Bacillus megaterium Strain That Persists in the Cotton Phyllosphere

Abstract

A lepidopteran toxin gene of the entomopathogen Bacillus thuringiensis subsp. kurstaki HD-1 was introduced into a cotton leaf-colonizing Bacillus megaterium strain, RS1, by conjugal transfer. Rifampin- and nalidixic acid-resistant colonies obtained after cell mating were screened for crystal production by microscopy. A transcipient, B. megaterium RS1-43, was selected by this procedure. Southern blot hybridization with both total DNA and HindIII-digested DNA of the transcipient showed positive signals with a cryIA-specific probe, suggesting the transfer of the lepidopteran-specific cryIA(a) gene. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis confirmed the presence of the 134-kDa toxic crystal protein specific to lepidopteran larvae in the transcipient. Survival studies with cultures of the transcipient at both vegetative and postvegetative growth stages on cotton, under field conditions, suggested that the bacterium persisted on the leaf surfaces for more than 28 days, with a gradual decline in the population level with time, while the donor, B. thuringiensis subsp. kurstaki, disappeared completely after 7 days following inoculation. An in situ differential crystal-staining technique indicated the production of crystals by the transcipient on cotton leaf surfaces for about 30 days. Leaf bioassays of cotton plants inoculated with a single spray of the transcipient showed 75- to 96% mortality to the first-instar larvae of Heliothis armigera up to 21 days, and this single spray conferred total protection to the plants for about 30 days by causing an antifeeding effect on the remaining larvae.