Genomic identification of Oryctes rhinoceros Nudivirus, a biocontrol agent of Coconut Rhinoceros Beetle

Coconut Rhinoceros Beetle (CRB, Oryctes rhinoceros) has its native range in Asia but is an introduced pest species of coconut palms in the Pacific. In an attempt to limit the damage and control the spread of CRB, Oryctes rhinoceros Nudivirus (OrNV) was introduced in the 1970s as a biocontrol agent. Following a period of very effective biocontrol there are now variants of CRB that are no longer affected by the OrNV biocontrol. We have initiated a study on the genetic diversity of CRB across the Pacific to address the question: are there CRB genotypes that are more susceptible to OrNV and/or more successful in establishing and spreading? In parallel we are studying OrNV isolates, originally identified in the CRB native range to determine the efficacy of different OrNV isolates as biocontrol agents. Here we describe the development of an assay for the rapid identification of different OrNV isolates. We have generated whole genome sequences of six OrNV isolates and, combined with OrNV genome sequences from the public domain, used this to design multiple PCR primer sets across the OrNV genome targeting regions of known variability. Currently, seven OrNV amplicons are generated for each sample. Individual samples are tagged with a unique barcode and combined for multiplex analysis using Oxford Nanopore Technologies sequencing. This assay allows us to detect and identify OrNV isolates in field samples from across the Pacific in order to understand and follow the presence and spread of strains across the region. In addition it assists in determining the efficacy of different OrNV isolates used in biocontrol assays where OrNV preparations are fed to CRB in the laboratory and in field settings. A detailed understanding of the diversity of OrNV isolates in the Pacific can contribute to improving management of CRB.