Molecular identification of Sitophilus spp. (Coleoptera Curculionidae) and detection of immature stages in wheat

Publication Type:Journal Article
Authors:A. Angiolillo, Boursier, L., Pilla, F., Germinara, G. S., Rotundo, G.
Date Published:2008
Type of Article:Article
:Sitophilus granarius, Sitophilus oryzae, Sitophilus zeamais

A polymerase chain reaction (PCR)-based assay is proposed to differentiate Sitophilus granarius (L.), S. oryzae (L.), and S. zeamais Motschulsky at different life stages and to detect their hidden infestation in wheat kernels. PCR amplification of genomic DNA using primers designed on the basis of the cytochrome oxidase subunit II gene sequence, followed by agarose gel electrophoresis, Successfully detected the immature stages (egg, larva, pupa) and adult of each species. DNA amplification resulted in a PCR product of 462 bp for S. granarius, whereas S. oryzae and S. zeamais yielded a 311 bp fragment. A sequence comparison between the latter species allowed to identify a mutation that was diagnostic between S. oryzae and S. zeamais through a PCR-RFLP protocol. Digestion of DNA resulted in an uncut fragment (3 11 bp) for S. oryzae, two fragments (174 bp + 137 bp) for S. zeamais and 3 bands (250 bp + 160 bp + 52 bp) for S. granarius. Multiplex PCR reaction enabled to differentiate the three species at all life stages also when simultaneously added to and ground with a 50 g sample of wheat kernels. The reproducibility of the results and high resolution as well as the technical simplicity of the developed molecular method demonstrated its usefulness in the diagnosis of Sitophilus spp. and detection of their hidden infestation in wheat.

Scratchpads developed and conceived by (alphabetical): Ed Baker, Katherine Bouton Alice Heaton Dimitris Koureas, Laurence Livermore, Dave Roberts, Simon Rycroft, Ben Scott, Vince Smith