Early detection and tracking of wood borers using improved environmental DNA aggregation and TaqMan quantitative PCR approaches in forests.

The increased number of invasive harmful organisms has exacerbated ecosystem damage. Early monitoring and timely control measures are urgently needed. Although environmental DNA (eDNA) application for monitoring aquatic organisms is well-established, its use in terrestrial monitoring remains limited, particularly for cryptic wood-borer pests like Monochamus alternatus, the primary vector of pine wilt disease. We developed and validated quantitative real-time polymerase chain reaction detection assays using TaqMan probes, targeting conserved regions of the ribosomal DNA (rDNA) internal transcribed spacer 1 and D2-D3 expansion segments of the 28S rDNA. eDNA surveys were conducted using the optimization of sample collection methods, effectively tracking M. alternatus in both laboratory and field environments. Laboratory tests revealed M. alternatus presence could be detected in emergence holes over 35 d, as well as in fresh oviposition scars from 1 to 7 d. The concentration of eDNA significantly decreased with the prolongation of sample storage time. Additionally, 3 novel eDNA aggregation approaches were developed to enhance detection sensitivity of wood-borer pests in forests: (i) rinsing aggregation, which gathers eDNA from the vicinity of emergence holes; (ii) wet cotton ball dipping, mainly used to collect residual eDNA in the oviposition scars and emergence holes; and (iii) natural predators, where M. alternatus can be identified by collecting ants on the surface of pine trunks. The results demonstrate the effectiveness and high sensitivity of the eDNA approach for M. alternatus monitoring. These findings provide valuable insights for early detection efforts and can serve as a reference for similar eDNA surveys targeting other wood-boring pests.