Non-destructive molecular methods to identify Monochamus alternatus (Coleoptera: Cerambycidae), a major vector of Bursaphelenchus xylophilus (Nematoda: Aphelenchoididae)

Abstract

Monochamus alternatus is one of the most important borers of conifers and the main vector of the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease. It causes massive death of pine trees and seriously affects the health of forest ecosystems. Traditionally, adults or larvae are identified by morphological or molecular methods. However, diagnosing M. alternatus larvae at different instars collected from forests is expensive and time-consuming. Non-destructive molecular diagnostic protocols are, therefore, being developed to detect biological traces (i.e. exuviae, excreta) and to determine the distribution and spread of this pest. In this study, based on the alignment of mitochondrial DNA cytochrome oxidase subunit I (COI) gene sequences of M. alternatus and other insect species, we designed the primer pair of Mal-SF/Mal-SR and probe of Mal-P for M. alternatus. TaqMan probe-based qPCR was developed to identify the occurrence of M. alternatus in forests by amplifying the DNA samples obtained from its adult, larva, frass, excreta and exuviae. The amplification results were very effective. The lowest amount of M. alternatus DNA that could be detected with a Cq of 31.93 in the mixed samples was 0.64 pg, showing very high sensitivity. This assay can easily identify M. alternatus from other non-target wood-borer species using its frass and exuviae, providing a new diagnostic protocol for monitoring the occurrence and distribution of M. alternatus in forests.