Nematodes of the genus Xiphinema (dagger nematodes) are migratory root ectoparasites with a broad host range. There are more than 260 species, of which approximately 60 belong to the Xiphinema americanum group (X. americanum sensu lato). Some of the species can specifically transmit certain nepoviruses of phytosanitary concern. Several subgroups of nepoviruses exist, each with their own specific vector species. Based on morphological and morphometrical data, the identification up to Xiphinema species level is quite difficult. Most of the time, it is impossible due to lack of specimens or appropriate nematode stadia. Currently, there is also a lack of molecular information of all Xiphinema species to develop reliable diagnostic tools such as PCR or barcoding. Direct damage caused by Xiphinema species is usually limited, the main issue is that some specimens are potential carriers of viruses. Whereas the whole X. americanum group had a quarantine status before the new Plant Health Legislation (December 2019), only 7 species retained this status and two species became regulated non-quarantine organisms. Still, their identification up to species level is very difficult. Focusing on a direct detection of these viruses in the nematodes could be an alternative to the difficult task of correctly identifying the Xiphinema species. This way, only specimens of the X. americanum group that are actually carrying viruses can be considered as a Plant Health risk, and consignments carrying Xiphinema spp. free of viruses could be allowed to enter trade. The aim of the project was to optimise a generic classic nepovirus detection test for nematodes of the genus Xiphinema and ultimately to validate it through the organisation of an interlaboratory test.

Based on literature, methods were selected to develop a diagnostic procedure for nepovirus detection in Xiphinema nematodes. Nematodes (X. diversicaudatum, X. index, and X. americanum s.s.) and nepoviruses (Arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV) and tomato ringspot virus (ToRSV)) were procured, virus transmission assays were carried out and the selected methods were compared and validated in the laboratory. This resulted in a recommended diagnostic protocol consisting of the Automated Zonal Centrifuge (AZC) for nematode extraction from soil, a method of choice for physical disruption of the isolated nematodes (slicing, bead beating and bead beating with collagenase pre-treatment) and the KingFisher MagMax Kit for RNA extraction. Due to inadequate sensitivity of generic nepovirus subgroup detection tests, nepovirus detection still relies on specific single nepovirus real-time PCR tests. The MinION nanopore technology was evaluated as an alternative for the future. MinION nanopore sequencing failed to detect GFLV and ToRSV in single nematode specimens. However, its potential was demonstrated by successful ArMV detection in six X. diversicaudatum individuals.