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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.

Success story of the Euphresco project 2018-A-293 'Phytosanitary risks of newly introduced crops (PRONC)'

Slide of the Euphresco project 2020-A-334 'Plant health status of Fagus spp. (FAGUSTAT)', Project funded through the Euphresco network

Slide of the Euphresco project 2019-A-330 'Detecting virus-carrying Xiphinema spp. as an alternative to Xiphinema identification up to species level in trade (XiphiVIR)', Project funded through the Euphresco network

Slide of the Euphresco project 2018-A-293 'Phytosanitary risks of newly introduced crops (PRONC)', Project funded through the Euphresco network

The project focussed on the optimization of monitoring strategies and pest management practices. In particular, the project aimed to optimize monitoring methods using attractant traps, to map the incidence for pine wood nematodes and possible vectors, to evaluate phenology and biology of relevant vectors and their pine wood nematodes relationship and to develop molecular identification tests for Monochamus spp. Participating research institutions included 7 research organisations from 5 countries; in four of these countries, the vectors were known to be present, in one country the PWN was known to be present and in one country no vector nor the nematode was known to be established., Report of the Euphresco funded project 'Focusing on Monochamus spp., insect vectors of Bursaphelenchus xylophilus (MONOCHAMUS)'

Various countries keep important nematode populations in (reference) collections for the purpose of research and identification. As funding has been scarce on projects concerning fundamental taxonomic research or the maintenance and accessibility of collections and archives, relevant collections cannot be managed optimally and maintained (i.e. indexing, updating) properly (see Euphresco Strategic Research Agenda). The main aim of this project was to gather together experts of nematode collections in different countries with the objective to make an inventory of live nematode collections in the participating countries: a list of the quarantine nematode populations hosted in the various countries was produced. Another objective was to exchange information on how nematodes are reared (maintenance and storage) in the different collections. Due to current legislation, in particular the Nagoya protocol and the new EU Plant Health Regulation, exchange of (reference) material is hampered severely: this has an impact on research collaboration and on the sustainability of reference collections. This point needs attention., Report of the Euphresco funded project 2016-F-186 'Inventory of living collections of cyst and root knot nematodes in Europe and their maintenance techniques (Cyst and Melo Collect)'

We still lack critical information on the spread of Meloidogyne spp. and especially the quarantine species M. chitwoodi and M. fallax in Europe and on the characteristics of the various populations. There is a need to standardize survey methods for detection of M. chitwoodi and M. fallax and to relate the current spread to parameters such as soil characteristics, climate and crop rotation practises. It is also important to know whether the current populations can be subdivided into different races or pathotypes. The initial objectives of the current study were 1) to perform a survey based on a standardized sampling protocol, 2) to monitor populations in the field for multiple years, and 3) to characterize M. chitwoodi and M. fallax populations using bioassays. Germany, France, Belgium, Turkey, Slovenia and the Netherlands participated in the project. Consensus on survey was lacking and the objectives 1 and 2 were abandoned. Only one country performed the envisioned survey as planned. Populations that had previously been detected in the participating countries and whose origin is not disclosed, were used for biological and molecular characterisation. Only M. chitwoodi populations were tested in the bioassay using a reference population M. chitwoodi called ‘Smakt’ and with prescribed plant material (differentials, according to Brown et al, 2009). The bioassay on Meloidogyne populations in four countries showed that it is difficult to stick to a uniform workflow despite of the presence of a standard protocol. Given these circumstances, we cannot be sure whether differences in host response are related to biological relevant differences or whether they result from differences in experimental procedures. However, the main question was to determine whether different races and/or pathotypes are present in the participating countries. This question was translated into the simple question of whether the populations differed from the reference population ‘Smakt’ on the set of differential host species per country. It remains debatable whether results on the response to Meloidogyne should be expressed in Pf-values or Pf/Pi values. The reference population reproduced on tomato, the susceptible potato cultivar and (to a lesser extent) on carrot. This population displayed Pf/Pi values While noting the shortcomings that arise from variation in the applied methods, we conclude that all tested populations from Europe most likely belong to race 1 of M. chitwoodi. The molecular characterisation of the different populations within Europe still has to take place, but the difficulty that the currently available markers do not show high levels of polymorphism are in agreement with the bioassay results. These findings are relevant for plant health management as control of these nematodes by using resistant crops, which is one of the few options, will become more straightforward. This underlines the need for crops that are resistant against M. chitwoodi from host race 1., Scientific report of the Euphresco funded project 'Population dynamics of Meloidogyne chitwoodi and M. fallax' (MELOPOP)

The aim of this Euphresco project was to coordinate ongoing and future research in the field of Meloidogyne enterolobii detection and identification and to validate new methods in a collaborative way. It contained six main topics: - Test performance study on nematode extraction methods to detect motile stages (second stage juveniles) of M. enterolobii in soil samples. - Test performance study on fast and simple DNA extraction from target nematode in complex nematode suspension after extraction from soil samples (Lysis buffer; Holterman et al., 2009). DNA needs to be clean enough (without inhibitors) for further by molecular detection/identification tools. Labs were encouraged but not required to use additional extraction methods. The aim was to test a fast and simple DNA extraction protocol that can be harmonized between NPPO laboratories in the future. - Methods development and in-house validation of qPCR methods to detect, identify and potentially quantify M. enterolobii in complex nematode suspensions obtained from soil samples (develop protocols for robust, specific and sensitive detection/identification that can be harmonized between NPPO labs, independent of lab equipment and chemistry). - Test performance study on protocols developed under topic 3. - Final meeting to communicate and discuss results. Optional workshop on Barcoding as new diagnostic tool for fast and reliable nematode identification was offered to interested groups. - Publication of results in peer-reviewed research journals and discussion for inclusion of protocols into EPPO diagnostic standard protocol. Identification of future research priorities in the field of Meloidogyne enterolobii research., Scientific report of the Euphresco funded project 'Development and validation of innovative diagnostic tools for detection and identification of the quarantine nematode Meloidogyne enterolobii in support of integrated plant protection strategies in the EU member states'

Potato cyst nematodes (PCN) Globodera pallida and Globodera rostochiensis cause serious damage to potato crops world-wide by restricting root growth and uptake of nutrients, leading to losses in tuber yield. They are considered quarantine organisms in many countries. In the EU, a control directive is active against these nematodes: the EU directive 2007/33/EC. The do’s and don’ts are described in this directive, especially for seed potatoes. This document also describes how the resistance level of potatoes against these nematodes has to be assessed before cultivars are allowed on the market and be used as a management tool. Resistant cultivars play the most important role in the management of PCN. The main goal of the project was to investigate if the consortium partners can identify the potato cyst nematode to species level and that the standard resistance testing method, described in the new control directive EU directive 2007/33/EC, can be performed. The aim of this project was twofold: to learn and validate test protocols for (1) identification and for (2) resistance testing., Scientific report of the Euphresco funded project 'Ring testing methods for identification and resistance testing' (Globodera)

Slide of the Euphresco project 'Ring testing of diagnostic methods for the identification of potato cyst nematodes (Globodera rostochiensis and G. pallida) and assessing resistance of potato cultivars' (GLOBODERA), Project funded through the Euphresco network