Abstract
The coconut rhinoceros beetle (Oryctes rhinoceros, CRB) is a serious pest of coconut and oil palms. It is native to South and Southeast Asia and was inadvertently introduced to Samoa in 1909. It has invaded many other Pacific countries throughout the last century. Oryctes rhinoceros nudivirus (OrNV), a natural pathogen of CRB in its native range, was successfully introduced as a classical biocontrol agent and has effectively suppressed invasive CRB populations for decades. However, resurgence of CRB has been recorded, with new invasions detected in several Pacific Island Countries and Territories. Additionally, new populations of CRB are emerging in some invaded areas that have a degree of resistance to the virus isolates commonly released for CRB biocontrol. Here, we designed a fast and reliable tool for distinguishing between different OrNV isolates that can help with the selection process to identify effective isolates for management of new CRB invasions. A comparison of 13 gene/gene region sequences within the OrNV genome of 16 OrNV isolates from native and invaded ranges allowed us to identify unique Single Nucleotide Polymorphisms (SNPs). With these SNPs, we developed an assay using multiplex PCR-amplicon-based nanopore sequencing to distinguish between OrNV isolates. We found that as few as four gene fragments were sufficient to identify 15 out of 20 OrNV isolates. This method can be used as a tool to monitor the establishment and distribution of OrNV isolates selected for release as biocontrol agents in CRB-infected areas.