Towards Improved Nepovirus Detection and Identification in Xiphinema Nematodes

Several Xiphinema nematode species are vectors of regulated nepoviruses. The aim of this study was to develop an optimized method to detect virus-carrying Xiphinema specimens. The study compared various techniques for nematode extraction from soil (automated zonal centrifuging and the Flegg modified Cobb method), nematode cuticle disruption (slicing, bead beating, and bead beating with collagenase), RNA extraction (RNeasy Plant Mini Kit, cetyltrimethylammonium bromide [CTAB], and KingFisher MagMAX RNA Isolation Kit), and nepovirus detection (nepovirus generic subgroup and species-specific quantitative and conventional reverse transcription polymerase chain reaction [RT-(q)PCR] assays, as well as shotgun sequencing with Oxford nanopore sequencing technology using a MinION device). Based on this comparison, a diagnostic procedure is proposed including the best-performing methods: nematode extraction using automated zonal centrifugation, physical disruption of nematode cuticle using bead beating or slicing, and RNA extraction using either the KingFisher MagMAX or the RNeasy Plant Mini Kit method. While existing generic nepovirus subgroup reverse transcription polymerase chain reaction (RT-PCR) assays lacked sensitivity, species-specific RT-(q)PCR assays successfully detected Arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), and tomato ringspot virus (ToRSV) in X. diversicaudatum, X. index, and X. americanum sensu stricto (s.s.), respectively. The reliability of nepovirus detection was higher with adults compared with juveniles. The minimum nematode quantity required varied depending on the specific nepovirus and the detection method used. [Figure: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.