A simple PCR-based approach for rapid detection of Ips typographus and Ips duplicatus in the presence of (associated) symbionts and parasites
In plant pest diagnosis, Sanger sequencing of marker genes (DNA-barcoding) is the most applied and appropriate method for the identification of insects. Standard PM7/129 of the European and Mediterranean Plant Protection Organization (EPPO) includes a number of primers and PCR protocols for diagnosing insect pests. LCO1490 and HCO2198 primers recommended herein were shown to be excellent tools for amplifying a fragment of the COI gene from a vast range of arthropods. The COI barcoding region is available for thousands of arthropod taxa in public databases and ready-to-use for evolutionary studies. However, we found that LCO1490 and HCO2198 primers are not working for bark beetles of genus Ips. The attempt to amplify this gene fragment from an individual organism using the barcoding primers led to DNA amplification of associated wasps and nematodes, which were apparently vectored by the beetle. Thus, new primers for Ips that bind specifically to another (non-barcoding) region of the COI gene were developed in the past years. These primers were successfully applied in phylogenetic analyses of this genus, resulting in the adverse effect that COI-based Ips phylogenies cannot be expanded to higher systematic categories without sequencing the outgroups (as they are not available in databases yet). Here we provide new primers for Ips that differ significantly from DNA sequences of Ips-associated wasps and nematodes and bind to a COI fragment that largely overlaps with the barcoding region proposed in the EPPO standard. Furthermore, using these primers we developed a quick PCR-based test for detecting Ips duplicatus, a quarantine pest currently emerging in many European countries.