Sporogenous Midgut Bacteria of the Leafroller,Pandemis pyrusana(Lepidoptera: Tortricidae)

Abstract

The composition of the microbial flora within the midgut of Pandemis pyrusana Kearfott was analyzed by using the polymerase chain reaction (PCR) and culturing techniques. Universal 16S ribosomal RNA gene-specific eubacterial primers were used in PCR, and the resulting products cloned and sequenced. High homology (86–95%) was obtained between the cloned products and Clostridium ramosum and Clostridium spiroforme when compared with computer database sequence information. Initial culturing steps aimed at endospore-forming bacteria yielded reproducible growth from a bacterium that matches most closely with Bacillus licheniformis when analyzed by partial 16S ribosomal DNA sequencing and biochemical/cultural profiling. This isolate was transformed with an Escherichia coli-Bacillus subtilis shuttle vector and reintroduced into the insect, where it remained stable under nonselective conditions for at least 9 d after initial ingestion. This confirmed the ability of the genetically altered bacterium to survive within the leafroller midgut in the midst of competing microorganisms and perhaps to serve as a suitable vehicle for biological control.