Abstract This document provides the conclusions of the pest survey card that was prepared in the context of the EFSA mandate on plant pest surveillance (M‐2020‐0114) at the request of the European Commission. The full pest survey card for Xiphinema rivesi (non‐EU populations) is published and available online in the EFSA Pest Survey Card gallery at the following link and will be updated whenever new information becomes available: https://efsa.europa.eu/plants/planthealth/monitoring/surveillance/xiphinema-rivesi

Plant-parasitic reniform nematodes of the genus Rotylenchulus are semi-endoparasites of herbaceous and woody plants occurring in regions with Mediterranean, tropical, and subtropical climates. In the present study, the occurrence of reniform nematodes in the rhizosphere of three olive orchards in Central Italy and six in Sicily (Italy) was investigated. Two Rotylenchulus species were recovered in olive groves in Central Italy, and no Rotylenchulus species were found in Sicily. Using the integrative taxonomy approach, combining morphological, molecular and multivariate morphological analyses, the two species were identified as R. borealis and R. macrodoratus. The D2-D3 sequencing of four individual specimens of Italian R. macrodoratus revealed the occurrence of unique haplotypes differing in nucleotide composition each other. Interestingly, the sequencing of different ITS clones from an individual specimen of the Italian R. borealis showed two ITS paralogs differing in length and nucleotide sequence compared with those of other specimens from the same population and showing higher similarity with those from other populations. Phylogenetic analyses, based on D2-D3 expansion domains of the 28S rRNA gene, ITS, and mitochondrial COI, confirmed the high level of ribosomal variability in both species and the occurrence of new mitochondrial haplotypes for the COI. The present study confirms the occurrence of high variability in Rotylenchulus genus and the existence of variant gene copies in the same specimen that could contribute to the survival of these species in different environments.

Surveys were conducted in the village of Shamgona in the Samegrelo Region of West Georgia, and two nematode species belonging to the Oscheius genus and a population of Heterorhabditis bacteriophora were isolated. Integrative diagnosis, combining morphology, PCA, sequencing and phylogeny, was carried for Oscheius sp. from the soil of hazelnut orchard. This population shows high similarity at morphological and morphometrical levels with O. cyrus from Iran, and the main distinctive features with O. cyrus from Iran are the presence of eight lateral lines and males. Sequence and phylogenetic analyses by using ITS, D2–D3 expansion domains of the 28S rRNA gene, the partial 18S rRNA gene, and the mitochondrial COI confirmed the identification as O. cyrus. A second population of Oscheius sp. and H. bacteriophora was characterized only at the molecular and phylogenetic level. The phylogenetic analyses, based on ITS and D2–D3 expansion domains, revealed the occurrence of O. insectivorus in Georgia grouping with O. insectivorus sequences with high support. The phylogenetic trees of H. bacteriophora, based on D2–D3 expansion domains, ITS, the partial 18S rRNA gene, and mitochondrial COI confirmed the high variability among geographical populations. The present study reports on the occurrence of two species of Oscheius in Georgia for the first time.

A nematode survey was carried out (in 2020) in apple and poplar orchards in Banja Luka, Bosnia-Herzegovina detected two nematode species belonging to the Xiphinema americanum-group. Polyphasic identification, combining morphological, molecular and phylogenetic analyses identified the nematodes as X. rivesi in apple orchards and X. incertum in poplar trees. Sequence and phylogenetic analyses used two rRNA genes (D2-D3 expansion segments of 28S rRNA, and ITS regions), and partial mitochondrial COI region detected high intra- and inter-specific variability within X. rivesi. These are the first reports of X. rivesi and X. incertum in Bosnia-Herzegovina, which extend the geographical distribution of these species in Europe.

Publications between 2000 and 2024 on plant pathogenic species of Helicotylenchus nematodes were reviewed using bibliometric analyses with VOSviewer software. Sequences in GenBank for the same period were used to reconstruct phylogenetic relationships and correctly assign all the Helicotylenchus sequences in the database. The Scopus database was selected for its professional standards, international visibility, broad coverage, and controlled-vocabulary thesaurus available for indexing and retrieving documents. GenBank collects nucleotide sequences and relevant bibliographic and biological annotations for sequence analyses. This study highlighted research trends and specific complexities of working with Helicotylenchus, and the suitability of molecular markers used for identification of the main pathogenic species and confirmation of occurrence of species complexes or cryptic species.

Summary The genus Oscheius includes species with different habits and some of them have been shown to kill some insect pests. The occurrence of Oscheius in hay meadow soils from the area recently invaded by the Japanese beetle Popillia japonica in Lombardy, Italy, has been surveyed. Oscheius myriophilus was detected at five out of 18 sites resulting positive to entomopathogenic nematodes (EPN), either alone or together with other genera of EPN, indicating a widespread occurrence. The morphological analysis of the strains retrieved showed that they are morphologically in agreement with those of the type population and with other populations recently described. The phylogenetic analysis supported the sub-grouping of all sequences of O. myriophilus from Lombardy with those present in the databases and a close relationship with O. safricana was observed. The capacity to kill the laboratory host Galleria mellonella was similar to that observed in a previous study related to isolates from P. japonica , although O. myriophilus did not cause significant mortality in laboratory trials with the invasive beetles. The isolates of O. myriophilus from Lombardy were characterised in relation to their infectivity to G. mellonella and response to environmental stress, such as heat tolerance and desiccation. Overall, O. myriophilus appears to be an important component of the EPN community of hay meadow soil in Lombardy and might play a role in the containment of the invasive P. japonica .

The occurrence of Xiphinema species in the rhizosphere of vineyards showing general decline phenotype in North Italy was investigated. Most of the collected soil samples contained two Xiphinema species belonging to X. americanum -group, along with X. index . Using the integrative taxonomy approach, combining morphological, molecular and multivariate morphological analyses, the two populations of the X. americanum -group were identified as X. rivesi and X. pachtaicum . Furthermore, one male specimen of X. rivesi was recovered and the presence of three juvenile developmental stages was confirmed based on the scatter diagram. D2-D3 expansion domains of LSU, ITS region and partial COI were used to determine the phylogenetic relationships of the two Xiphinema species from North Italy with other species belonging to X. americanum -group. Five other Xiphinema populations from different geographical areas were included in this study and only molecularly identified as X. rivesi, X. pachtaicum and X. incertum by sequencing the ITS and D2-D3 expansion domains of LSU rDNA.

Once a nematode has been identified, to conduct studies for screening programs or pathogenicity tests, it is necessary a supply of large numbers of nematodes from field crops or reproduced and stored to be used in periods of the year when they are not available from fields. Therefore, nematodes must be reared in greenhouse or under in vitro conditions and stored for future needs. In this chapter, suggestions are given on how to obtain nematodes from fields and reproduce most of them on host plants in greenhouse (mainly Meloidogyne spp. and Globodera spp.) or in vitro. Reproductions in vitro include: On suitable callus of host plants (Pratylenchus spp., Ditylenchus spp.) On fungal cultures mainly of Botrytis cinerea or Alternaria spp. for Aphelenchoides spp. and other aphelenchids, including Bursaphelenchus xylophilus. On carrot disks for Pratylenchus spp. and Ditylenchus spp. Other specific media, such as garlic, potato, and sweet potato for D. destructor, and cocoyam disks for Radopholus similis. Guidelines are also given to store different nematodes for rather long times, including in vitro methods and in infected seeds, hay, and other plant parts. No information is given on how to prepare and store fixed materials.

A population containing specimens belonging to Anatonchus genus collected along the edge of irrigation canals from Filippiada, Epirus (N.W Greece) is described and illustrated. The predatory ability and type of feeding against phytoparasitic free-living nematodes and larval specimens of insects (Curculionoidea: Coleoptera) are also showed. All observations were based on direct analysis of the gut contents in glycerine infiltrated specimens. The feeding type of its trophic activity was characterized by engulfing whole and intact prey, while in cases of large size nematode prey or larvae of insects, the piercing and content-sucking feeding type was observed. Several entire tylenchid and/or dorylaimid nematode species were observed in the digestive tract of Anantonchus sp. (75%). No evidence of preference for selecting its prey species was observed. Key Words: Greece, Mononchida, Predation, Potential Biological Control, Taxonomy

During a nematode survey in Iran, an abundant population of sheathoid, migratory, root-ectoparasitic nematodes was recovered from a tea, Camellia sinensis (L.), Kuntze plantation for the first time. Morphological and molecular characterization identified the Iranian population as Hemicriconemoides kanayaensis. The morphometrics of H. kanayaensis agreed with the original description. Phylogenetic relationships within Hemicriconemoides-based on ITS region, D2 to D3 expansion regions of the 28S rRNA, and the partial 18S rRNA genes along with the partial mitochondrial COI gene-confirmed the occurrence of H. kanayaensis on the tea plantation in Iran. Principal component analysis (PCA) confirmed the high intraspecific and interspecific variabilities among Hemicriconemoides species and between H. kanayaensis populations.

The term "microbial control" has been used to describe the use of microbial pathogens (bacteria, viruses, or fungi) or entomopathogenic nematodes (EPNs) to control various insect pest populations. EPNs are among the best biocontrol agents, and major developments in their use have occurred in recent decades, with many surveys having been conducted all over the world to identify EPNs that may have potential in the management of insect pests. For nematodes, the term "entomopathogenic" means "causing disease to insects" and is mainly used in reference to the bacterial symbionts of Steinernema and Heterorhabditis (Xenorhabdus and Photorhabdus, respectively), which cause EPN infectivity. A compendium of our multiannual experiences on EPN surveys and on their collection, identification, characterization, and use in agro-forestry ecosystems is presented here to testify and demonstrate once again that biological control with EPNs is possible and offers many advantages over chemicals, such as end-user safety, minimal damage to natural enemies, and lack of environmental pollution, which are essential conditions for an advanced IPM strategy.

The nematicidal activity of garlic extracts is known on root–knot nematodes but never investigated on the grapevine nematode Xiphinema index. In this study, the nematicidal activity of a commercial garlic extract formulate (GEF) was assessed on X. index, both in vitro and in a pot assay. In the in vitro assays, mixed specimens of X. index were exposed to a 0–4 mL L−1 range of GEF concentrations, checking nematode immotility and mortality after 2, 4 or 8 h. In the experiments on potted grapevines, plants cultivated in soil infested by X. index were irrigated twice at a 15-day interval with 0.05, 0.2 and 0.5 mL L−1 solutions of GEF, including nontreated soil as a control. An almost complete mortality of X. index specimens occurred after a 2 h exposure to a 2 mL L−1 GEF concentration, while an 8 h exposure to even the 0.0312 and 0.0156 mL L−1 solutions resulted in about 50% and 30% mortality, respectively. Soil treatment with a 0.5 mL L−1 GEF solution significantly reduced the population of X. index and increased the grapevine root growth compared to nontreated soil or soil treated with the lower dosages. Results of this study indicated that garlic-based nematicides could be an effective tool for X. index management in organic and integrated vineyards.

Plant parasitic nematodes are a serious threat to crop production worldwide and their control is extremely challenging. Fungal volatile organic compounds (VOCs) provide an ecofriendly alternative to synthetic nematicides, many of which have been withdrawn due to the risks they pose to humans and the environment. This study investigated the biocidal properties of two fungal VOCs, 1-Octen-3-ol and 3-Octanone, against the widespread root-knot nematode Meloidogyne incognita. Both VOCs proved to be highly toxic to the infective second-stage juveniles (J2) and inhibited hatching. Toxicity was dependent on the dose and period of exposure. The LD50 of 1-Octen-3-ol and 3-Octanone was 3.2 and 4.6 µL, respectively. The LT50 of 1-Octen-3-ol and 3-Octanone was 71.2 and 147.1 min, respectively. Both VOCs were highly toxic but 1-Octen-3-ol was more effective than 3-Octanone. Exposure of M. incognita egg-masses for 48 h at two doses (0.8 and 3.2 µL) of these VOCs showed that 1-Octen-3-ol had significantly greater nematicidal activity (100%) than 3-Octanone (14.7%) and the nematicide metham sodium (6.1%). High levels of reactive oxygen species detected in J2 exposed to 1-Octen-3-ol and 3-Octanone suggest oxidative stress was one factor contributing to mortality and needs to be investigated further.

The recent invasion of the Japanese beetle Popillia japonica Newman (Coleoptera: Scarabaeidae) in northern Italy offered the opportunity to explore the entomopathogenic nematodes (EPNs) associated with the soil of hay meadows. A total of 61 sites were considered for nematode sampling, and from 17 of them (27.9%) EPNs were isolated and further characterized with molecular and morphological techniques as well as with laboratory bioassays. Two main species, Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) and Steinernema carpocapsae Weiser (Rhabditida: Steinernematidae) were recorded with the same frequency together with a few other species (Rhabditida: Oscheius sp. and Phasmarhabditis sp.). The newly isolated EPN populations were characterized for their activity (penetration rate) and infectivity to P. japonica grubs. EPNs occurrence was related to the time of beetle invasion at each sampling site and there was evidence of a density‐dependent response of the EPNs community to P. japonica density. The invasion of P. japonica apparently did not significantly affect the occurrence of native grubs, although a tendency to a decline was observed and should be further investigated.

Since the first detection of Meloidogyne graminicola in Piedmont, North Italy, in 2016, further inspections for the presence of the rice root-knot nematode were carried out in rice fields of neighboring regions, in accordance with the Italian NPPO (National Plant Protection Organization) to support the official phytosanitary measures, to enable the early detection of the rice pest, and to prevent its spread within the national territory. In 2018, surveys of rice fields in Lombardy region revealed a new occurrence of M. graminicola. In the present study, we confirmed the identification of the rice nematode in Lombardy using the ribosomal ITS region and the mitochondrial COI and COII genes. The sequences and phylogenetic analyses revealed that Lombardy M. graminicola population grouped in all trees in the main cluster containing Meloidogyne species belonging to graminis group, but always in a different subgroup compared to the Piedmont population of M. graminicola. These results clearly suggest that the two Italian populations have been recently and independently introduced and confirm that the geographic origin is not the main factor leading to M. graminicola population variability.

During the inspection of vegetable material for EXPO 2015 in Milan (Italy), samples of bark from an unidentified coniferous tree were analysed and screened for the presence of the pinewood nematode, Bursaphelenchus xylophilus, in accordance with the surveillance of European Union to enable early detection and prevent further spread. In this study, nematodes associated with the bark of unidentified coniferous tree were collected and identified by morphometrics and sequencing of ITS, D2-D3 expansion domains of the 28S rRNA and the partial 18S rRNA genes. The sequences obtained and the phylogenetic analyses, using the ITS, D2-D3 expansion domains of the 28S rRNA and the partial 18S rRNA genes, revealed the presence of four Aphelenchidae species. Two of the species recovered were phylogenetically related to Cryptaphelenchus species. One of the two was identified as C. minutus, as it showed morphology and morphometry largely overlapping with Bursaphelenchus minutus, but phylogenetically closely related to Cryptaphelenchus species. The other three species recovered were only molecularly identified herein, by sequencing the ITS and the D2-D3 expansion domains, as Cryptaphelenchus sp. 1 and two Aphelenchoides species.

FLOTAC Techniques have been widely acknowledged as an effective method for the extraction of human and animal parasites. The present study is the first application of FLOTAC basic technique (FBT) for the extraction of phytoparasitic nematodes from soil and infested plant roots. Eggs and second stage juveniles (J 2 ) of the root-knot nematode Meloidogyne incognita were extracted from infested soil and tomato roots either by FBT and conventional nematode extraction methods, such as centrifugal flotation and root maceration techniques, respectively. The number of M. incognita J 2 and eggs extracted from soil by FBT was always significantly higher compared to the extraction with the centrifugal flotation method, averaging 277 vs 35 eggs and J 2 mL -1 soil. Conversely, no significant differences were observed between FBT and the root maceration technique in the extraction of eggs and J 2 from tomato roots. Results demonstrated that FBT can be highly effective also for the extraction of phytoparasitic nematodes. Due to its accuracy and sensitivity, FBT seems particularly suitable for nematode surveys in wide geographical areas, where an accurate and rapid detection of present phytoparasitic nematofauna is required.

Summary Phasmarhabditis thesamica n. sp., belonging to the family Rhabditidae, was isolated from the bodies of the invasive slug, Deroceras reticulatum in Georgia. Nematodes of this genus have never been recorded previously in Georgia. Phasmarhabditis thesamica n. sp. was characterised at morphological, morphometrical and molecular level. Females are 2052 (1863-2241) μm long, lateral fields consisting of three protruding central ridges and four incisions. The vulval opening is in the middle of the body. The tail is conical, long, tapering to a filiform tip, with prominent rod-shaped phasmids. The reproductive system is didelphic-amphidelphic. Males are 1301 (1123-1456) μm long, with an open peloderal bursa, which is supported by nine pairs of genital papillae 1 + 1 + 1/2 + 1 + 3, and two phasmids located close to tail tip. The molecular phylogeny of P. thesamica n. sp. inferred by using ITS, D2-D3 expansion domains of the 28S rRNA gene and the 18S rRNA gene revealed close relationships with P. clausiliiae, with high support.

Summary Two populations of needle nematode, Longidorus longicaudatus Siddiqi, 1962, are described from Quercus hemisphaerica, Q. nigra and Q. virginiana from north Florida, USA. These populations are characterised morphologically by females having a body shorter than 3800 μm, a rounded or slightly flattened lip region, an amphidial fovea pouch-like often with two symmetrical lobes, an odontostyle 99-110 μm long, a conoid tail ending in a bluntly pointed terminus, ranging values of ratio c′ greater than 2, and males very rare. The polytomous code for these populations is A34, B23, C2, D23, E2, F12, G12, H6, I12, J1, K6. Although the morphology and morphometrics of these two populations fit the original description of Longidorus longicaudatus, Florida specimens have greater diameters of lip region, mid and anal body than those of the five type specimens used for the description of this species. The Florida L. longicaudatus is similar to L. paralongicaudatus, but differs from the paratypes of this species in having smaller and greater values of ratios c (53.8 (43.8-64.5) vs 79.2 (61.9-103.5)) and c′ (2.4 (2.1-2.9) vs 1.8 (1.5-2.0)), respectively, and longer tail (60 (53-67) vs 46 (36-53) μm). Molecular characterisation of one of the two Florida L. longicaudatus populations was made based on the D2-D3 of 28S rRNA, ITS1 rRNA and COI gene sequences. The results of the ITS1 rRNA gene sequence analysis indicated that it is genetically different from L. paralongicaudatus. A few specimens of a needle nematode associated with L. longicaudatus were identified morphologically and molecularly as the pine needle nematode, L. americanus. This detection is a new record of the occurrence of the pine needle nematode in Florida.