A simple PCR-based approach for rapid detection of Ips typographus and Ips duplicatus in the presence of (associated) symbionts and parasites

Abstract

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.

The influence of storage conditions and total DNA extraction protocol on the results of molecular analysis of the European spruce bark beetle (Ips typographus L.)

“…However, host-specific primers are more useful for molecular identification in the presence of endoparasites. As previously proposed (Becker et al, 2021), the results demonstrate the limitations of using universal primer pairs such as LCO1490/HCO2198 with some arthropod samples. This approach might work for other Scolytinae taxa without the problems of non-target amplification, as demonstrated by Marinč et al (2019).…”

“…We tested two different sets of primers targeting the arthropod COI gene with an end-point PCR. The LCO1490/HCO2198 primer pair was developed by Folmer et al (1994) and the Ips_F_cons/Ips_R_cons pair was developed by Becker et al (2021) (Table 2).…”

“…The calculated average amount with the standard deviation of total DNA extracted per gram of input (Folmer et al, 1994;EPPO, 2021;Becker et al, 2021) Preglednica 2: Začetni oligonukleotidi in pogoji, uporabljeni v raziskavi (Folmer et al, 1994;EPPO, 2021;Becker et 3). It was demonstrated that there was a significant difference between the amount of DNA extracted per gram of input specimen among the three assessed protocols, i.e., P1, P2, and P3 (Table 4).…”

Molecular identification of Anoplophora glabripennis (Coleoptera: Cerambycidae) and detection from frass samples based on real-time quantitative PCR

“…Therefore, a molecular diagnostic tool to confirm A. glabripennis infestation through the analysis of frass material collected on the outside of the plant can be an effective, non-invasive complement to traditional detection methods. Using the DNA barcoding primers LCO1490 and HCO2198 (Folmer et al 1994), recommended in PM 7/129 (EPPO 2016, for frass material leads to unspecific co-amplification of eukaryotic genomic DNA from the wood matrix (Becker et al 2020). Although this is not surprising as primers were originally designed for barcoding as many eukaryotes as possible, it turned out to be misleading while working on samples containing different DNA sources, such as A. glabripennis frass.…”