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The Mediterranean olive cyst nematode, Heterodera mediterranea, is a pest of olive trees that is found in Spain, Italy, and Tunisia. In this paper, conventional and real-time PCR assays for the identification of H. mediterranea was developed. The hsp90 gene fragment was used to design a species-specific primer and a TaqMan probe. The specificity of the primer/probe set was tested in singleplex or multiplex reactions against Mediterranean olive cyst nematode and non-target nematodes. In multiplex real-time PCR experiments with the specific and universal primer/probe sets, fluorescent signals were simultaneously monitored for hsp90 and D3 of 28S rRNA target genes. The results of the experiments showed that the assays with species-specific primer/probe were sensitive enough to detect the DNA from 0.03 of a second-stage juvenile (J2) H. mediterranea in a reaction tube. [ABSTRACT FROM AUTHOR]

Needle nematodes have an economic importance by causing damage to a wide range of natural and cultivated plants not only by directly feeding on root cells, but also by transmitting plant nepoviruses. Two new Longidorus nematodes, Longidorus oakcrassus n. sp. and Longidorus oakgracilis n. sp., are described and illustrated from populations associated with the rhizosphere of Pyrenean oak (Quercus pyrenaica Wild.) in southern Spain. The taxonomic position of both new species within the genus was assigned using an integrative approach. Morphologically, L. oakcrassus n. sp. is characterized by a female with a large and robust body size (9.2–12.2 mm), lip region anteriorly flattened to slightly rounded and almost continuous or slightly offset by a depression with body contour, ca 25.5–32.0 μm wide, amphidial fovea with slightly asymmetrical lobes, stylet composed by an odontostyle moderately long (110.0–133.5 μm) and odontophore weakly developed, pharynx short ending in a terminal pharyngeal bulb with common arrangement of pharyngeal glands, tail short almost hemispherical shape. Longidorus oakgracilis n. sp. is characterized by having a moderately long and thin female body (5.4–7.9 mm in length), a bluntly-rounded lip region, set off from body contour by a slight depression, amphidial fovea funnel-shaped without lobe, odontostyle moderately long (94.0–106.0 μm), pharyngeal bulb with common arrangement of pharyngeal glands, short tail, bluntly hemispherical. The presence of males is common in both species. Integrative diagnosis was based on molecular data using D2-D3 expansion domains of the 28S rRNA, 18S rRNA, ITS1 rRNA and partial coxI gene sequences and morphology. Although different gene markers show variations in the phylogenetic relationships, phylogeny indicated that L. oakcrassus n. sp. is phylogenetically related with several species described from the Iberian Peninsula, including L. oakgracilis n. sp., which is clustered with L.cf. olegi, L. lusitanicus and L. silvestris. [ABSTRACT FROM AUTHOR]

A new and a known longidorid nematode, Paralongidorus lusitanicus n. sp. and Paralongidorus plesioepimikis, are described and illustrated from populations extracted from soil associated with grapevine (Vitis vinifera L.) from Escaroupim and Pó (central-Western Portugal), respectively. The new needle nematode P. lusitanicus n. sp. is characterised by a very large body size (8072-12,022 μm), an expanded and rounded lip region, ca 30 μm wide, with a clear constriction followed by a depression posterior to the amphidial aperture, amphidial fovea very large (11.0-19.0 μm), stirrup-shaped, with conspicuous slit-like aperture as shown in scanning electron microscopy studies, a very long and flexible odontostyle (180.0-223.0 μm), guiding ring located at 28.0-41.5 μm from anterior end, vulva anterior to the mid-body (34-41%), a dorsally convex-conoid tail with rounded terminus (29-42 μm long), bearing two or three pairs of caudal pores and males common (ratio 1:1.6 females) with spicules ca 80 μm long. Morphological and morphometric traits for P. plesioepimikis fit well with the original description, and is reported for the first time in Portugal. Integrative diagnosis of both species was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA and partial 18S-rDNA. The phylogenetic relationships of these species with other Paralongidorus spp. using these three molecular markers indicated that P. lusitanicus n. sp. clustered together with other Paralongidorus spp. forming a sister clade with P. plesioepimikis, both of them sharing a large body, long odontostyle, an anteriorly located vulva and an expanded and rounded lip region with a clear constriction followed by a depression posterior to the amphidial aperture. [ABSTRACT FROM AUTHOR]

Cyst nematodes obtained from commercial carrot fields in Ontario (Canada) and northern and southern Italy were subjected to morphological and molecular examination. Morphology of cyst cone tops, males and second-stage juveniles (J2) indicated the nematode species was the Carrot Cyst Nematode (CaCN), Heterodera carotae. The sequence of the Internal Transcribed Spacer (ITS), D2-D3 region of the 28S gene of ribosomal RNA, cytochrome oxidase I of mitochondrial DNA (coxI), and a heat shock protein gene (hsp90), from single cysts were also examined. Sequences of ITS and D2-D3 placed all the nematodes with Heterodera carotae and other Heterodera spp. belonging to the Goettingiana group in the same clade. The novel nine coxI sequences obtained also clustered in a well-supported phylogenetic clade for H. carotae. Similarly, the six new hsp90 sequences of H. carotae generated in this study were placed in a well-supported clade (PP = 1.00) together with other two sequences of H. carotae from Greece. Restriction Fragment Length Polymorphism (RFLP) of ITS-PCR products gave a restriction pattern for RsaI different than H. carotae but the other 6 restriction patterns were similar as described in former research. A diagnostic conventional PCR method was developed based on a primer set to be specific for H. carotae using coxI sequence. These primers were also used in real time PCR to generate a melt curve specific to H. carotae. Limit of detection for CaCN in conventional PCR reaction was a single J2. [ABSTRACT FROM AUTHOR]

The spiral nematode Helicotylenchus oleae is an ectoparasite of olive roots and is distributed in some countries in the Mediterranean Basin. In this study, we provided morphological and molecular characterisation of topotypes from southern Italy, as well as of several other populations from Crete (Greece) and Spain. Correct identification of plant-parasitic nematode species is essential to establish appropriate control strategies and for preventing their spread to other areas. Helicotylenchus oleae is reported for the first time in Greece (Crete). Integrative morphometric and molecular data for H. oleae populations using D2-D3 expansion segments of 28S rDNA, ITS-rDNA, and the cytochrome c oxidase subunit 1 (coxI), were in agreement with the original descriptions of the species, except for some minor differences, which may be a result of intraspecific variability. The phylogenetic relationships of this species with other representatives of Helicotylenchus spp. using D2-D3 expansion segments and the ITS-rRNA region was studied showing a low intraspecific diversity for H. oleae species. For the first time, coxI molecular data is obtained for the genus Helicotylenchus [ABSTRACT FROM AUTHOR]

The reniform nematodes of the genus Rotylenchulus are semi-endoparasites of numerous herbaceous and woody plant roots and distributed in regions with Mediterranean, subtropical and tropical climates. In this study, we provide morphological and molecular characterisation of three out of 11 valid species of the genus Rotylenchulus: R. macrodoratus, R. macrosoma, and R. reniformis from Greece (Crete), Italy and Spain. The overall prevalence of reniform nematodes in wild and cultivated olives in Greece, Italy, and Spain was 11.5%, 19.0% and 0.6%, respectively. In Greece, R. macrodoratus and R. macrosoma were detected in cultivated olive with a prevalence of 8.2% and 6.2%, respectively, but none of them were found in wild olive. This is the first report of R. macrosoma in Greece. Only one reniform nematode species was detected in olive from Italy and Spain, viz. R. macrodoratus and R. macrosoma, respectively. The parasitism of R. macrosoma on hazelnut in northern Spain was also confirmed for the first time. This study demonstrates that R. macrodoratus and R. macrosoma have two distinct rRNA gene types in their genomes, specifically the two types of D2-D3 for R. macrosoma and R. macrodoratus, the two types of ITS for R. macrodoratus and the testing of the ITS variability in other R. macrosoma populations in different countries. Rotylenchulus macrosoma from Greece and Spain showed differences in nucleotide sequences in the ITS region and D2-D3 of 28S rRNA gene. [ABSTRACT FROM AUTHOR]

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276-4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50-54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c' ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S-rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 ( coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae. [ABSTRACT FROM AUTHOR]

Pseudomonodelphic dagger nematodes of the genus Xiphinema are characterized by having one of the genital branches reduced and lacking an ovary. They are usually reported from tropical regions. Nematode surveys conducted during rainy seasons in Costa Rica resulted in detection of several Xiphinema costaricense populations, but also other pseudomonodelphic and didelphic species of Xiphinema. We undertook detailed integrative morphometric and molecular studies using D2-D3 expansion segments of 28S rDNA, and ITS1-rDNA. From those studies, we also identified several populations of Xiphinema krugi and two populations of Xiphinema setariae with characteristics in agreement with those of the original and later descriptions of these species. The phylogenetic analyses of these species with other representatives of Xiphinema spp. indicated that pseudomonodelphic species are phylogenetically related ( X. costaricense and X. krugi). On the basis of ITS1 sequences of X. costaricense and X. variegatum from Brazil, as well as similar morphology and morphometrics of both species, the latter is proposed here as a junior synonym of the former. Also, our morphometric data showed some intraspecific variability within X. setariae, which in combination with the molecular evidence, suggests that X. setariae and X. vulgare need to be considered as a single taxon. [ABSTRACT FROM AUTHOR]

The occurrence and geographic distribution of longidorid nematode species inhabiting the rhizosphere of cultivated olive (cvs. Chemlali and Chétoui) in Tunisia were investigated. Morphological and morphometrical studies identified three Longidorus and six Xiphinema species, with frequencies of prevalence as following: Longidorus africanus (23.0 %), L. euonymus (4.5 %) , L. glycines (13.7 %) , Xiphinema conurum (13.7 %) , X. italiae (36.4 %), X. meridianum (13.7 %), X. pachtaicum (18.2 %), X. robbinsi (9.1 %), and Xiphinema sp. (4.5 %). The three Longidorus species were reported for the first time in Tunisia, in addition to two species of Xiphinema (viz. X. meridianum and X. robbinsi). Molecular characterisation using D2-D3 expansion regions of 28S rRNA and ITS1-rRNA was carried out and Bayesian inference analysis was used to reconstruct phylogenetic relationships among these species and with other longidorids. Twenty-five new D2-D3 of 28S rRNA gene sequences were obtained in the present study, seven for Longidorus and 18 for Xiphinema spp., as well as 14 new ITS1 rRNA gene sequences (seven for Longidorus and seven for Xiphinema spp.). [ABSTRACT FROM AUTHOR]

Correct identification of Longidorus species in Costa Rica is essential to establish appropriate control strategies for preventing the spread of these nematodes. Nematode surveys conducted in the rainy seasons from 2013 to 2015 in areas arbitrarily chosen and widely distributed in the whole territory of Costa Rica, resulted in an overall prevalence of Longidorus spp. infesting soils both cultivated, ornamental and wild plants of 40.26 %. Integrative morphometric and molecular data for Longidorus populations were obtained using D2-D3 expansion segments of 28S rRNA, ITS1-rRNA, and the partial 18S-rRNA, identifying a solely species identified as Longidorus laevicapitatus. Morphology and morphometrical traits analysis of these populations of L. laevicapitatus were in agreement with those of the original and posterior descriptions of the species, except for some minor differences, which may be a result of intraspecific variability. The phylogenetic relationships of this species with other representatives of Longidorus spp. using D2-D3 expansion segments and the partial 18S indicated that L. laevicapitatus clustered clearly separately in a basal position in both phylogenetic trees. [ABSTRACT FROM AUTHOR]

A new longidorid nematode, Longidorus persicus n. sp., is described and illustrated from a population extracted from soil associated with wild rose ( Rosa sp.) naturally growing in the mountains close to the village of Cheshmeh-e-Nezamei near the city of Gilan-e-Gharb, Kermanshah province, western Iran. The new needle nematode is characterised by a large body size (6550-7763 μm), an anteriorly flattened lip region, ca 13 μm wide, distinctly set off from body contour by a depression, amphidial fovea large, pocket shaped, slightly asymmetrically bilobed, a moderately long and flexible odontostyle ca 86 μm long, stylet guiding ring located at ca 25 μm from anterior end, posterior arrangement of the pharyngeal gland nuclei, vulva almost equatorial (49-54 %), tail short, about 3/4 of its width, dorsally convex-conoid, with rounded terminus, with a c' ratio ca 1.3, bearing three pairs of caudal pores and male rare (ratio 1:10 females) with spicules ca 46 μm long. Integrative diagnosis was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, and partial 18S-rDNA. The phylogenetic relationships of this species with other Longidorus spp. using D2-D3 expansion segments and partial 18S-rDNA indicated that L. persicus n. sp. clustered together with L. perangustus and L. euonymus: both of them sharing an anteriorly flattened lip region and distinctly set off from body contour by a depression. [ABSTRACT FROM AUTHOR]

A new and a known species of Rotylenchus from Crete, each collected from the rhizosphere of olive, are described and illustrated based on morphological, morphometric and molecular studies. Rotylenchus cretensis n. sp. is characterised by having a truncate lip region with irregular longitudinal striation, lateral field areolated only in pharynx region, stylet length less than 41 μm, vulva located at 53-59 %, conoid-rounded to dorsally convex-conoid female tail with 7-11 annuli and specific D2-D3 of 28S, ITS, and partial 18S rRNA. Rotylenchus cretensis n. sp. appears to be similar to R. urmiaensis, having overlapping character ranges. Rotylenchus cretensis is also similar to R. striaticeps from which has got shorter stylet length (35.5-41.5 vs 39-50 μm), female tail shape (conoid-rounded to dorsally convex-conoid vs rounded), shorter spicules length (34-40 vs 41-50 μm), and phasmid position (varying from at level to three annuli posterior to the anus vs at level to seven annuli anterior to anus). Multivariate analysis of variance performed on R. cretensis n. sp., as well as two populations of R. striaticeps and R. urmiaensis from Iran on combined morphometric characters data set revealed a significant effect by species populations, indicating that closely morphological and morphometrically species differed significantly from each other. Additionally, analysis of variance showed that morphometric characters such as lip region width, distance from phasmid to tail tip (PHS) or a ratio can be used as key morphometric characters for species differentiation in this group. The results of phylogenetic analyses based on D2-D3 region, ITS of rRNA, and the partial 18S sequences confirmed the close molecular relationships between R. cretensis n. sp ., R. urmiaensis and R. striaticeps, but clearly support the proposal of R. cretensis n. sp. as a new species. Also, the recognition of this cryptic species within Rotylenchus shows that the biodiversity of these nematodes is still not fully understood and requires further studies. Additionally, R. cypriensis is described and molecularly analysed for first time in Greece. [ABSTRACT FROM AUTHOR]

The camellia root-knot nematode Meloidogyne camelliae was detected in Italy parasitizing roots of Japanese camellia ( Camellia sasanqua) flowering bonsai trees imported from Japan, and it represents a new record for Europe. Morphology and morphometrical traits analysis of the intercepted population of M. camelliae were in agreement with those of the original description of the species, except for some minor differences in second-stage juvenile stylet length and c ratio, spicules and gubernaculum in males, which may be a result of intraspecific variability. Integrative molecular data for this species were obtained using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, and the protein-coding mitochondrial gene COI. The phylogenetic relationships of this species with other representatives of Meloidogyne spp. using D2-D3 expansion segments and ITS1 indicated that M. camelliae clustered together with M. artiellia and M. baetica. Histological observations of C. sasanqua feeder roots infected by M. camelliae reveal slight swellings of the root feeding sites, and most egg-laying females protruding from the root. The anatomical alterations at feeding sites consist of 3-8 hypertrophied, polinucleate giant cells/female, but no hyperplasia was induced unlike what commonly occurs for most members of the genus. [ABSTRACT FROM AUTHOR]

The Japanese cyst nematode Heterodera elachista was detected parasitizing corn cv Rixxer in Bosco Mesola (Ferrara Province) in Northern Italy. The only previous report of this nematode was in Asia (Japan, China and Iran) attacking upland rice; being this work the first report of this cyst nematode in Europe, and confirmed corn as a new host plant for this species. Integrative morphological and molecular data for this species were obtained using D2-D3 expansion regions of 28S rDNA, ITS1-rDNA, the partial 18S rDNA, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I ( COI), and the heat-shock protein 90 ( hsp90). Heterodera elachista identified in Northern Italy was morphologically and molecularly clearly separated from other cyst nematodes attacking corn (viz. H. avenae, H. filipjevi, H. delvii, H. oryzae, H. sacchari, H. sorghi, H. zeae, Punctodera chalcoensis, and Vittadera zeaphila) and rice ( H. oryzae, H. sacchari). The phylogenetic relationships of H. elachista from Northern Italy with other cyst-nematodes using rDNA and mtDNA showed a separation of the genus Heterodera in various morphospecies groups based on vulval cone structures. The development and parasitic habit of H. elachista on naturally infected corn cv Rixxer confirmed a typical susceptible reaction, including multinucleate syncytial cells in parenchymatic cells. Under greenhouse conditions, H. elachista successfully reproduced on two crops widely used in Northern Italy, such as corn (cv PR 33) and rice (cv Baldo). Considering the limited host-range of this nematode, that include two of the three world's most important crops, special attention is needed for avoiding the dispersal of this nematode into new areas, by movement of soil on equipment, water, and contaminated containers infested soil, or agricultural practices. [ABSTRACT FROM AUTHOR]

The non-cyst forming heteroderid nematode Cryphodera brinkmani was detected in Italy parasitizing roots of Japanese white pine bonsai ( Pinus parviflora) trees imported from Japan. Morphology and morphometrical traits of the intercepted population on this new host for C. brinkmani were in agreement with the original description, except for some minor differences on male morphology. Integrative molecular data for this species were obtained using D2-D3 expansion regions of 28S rDNA, ITS1-rDNA, the partial 18S rDNA, and the protein-coding mitochondrial gene, cytochrome oxidase c subunit I ( COI). The phylogenetic relationships of this species with other representatives of non-cyst and cyst-forming Heteroderidae using ITS1 are presented and indicated that C. brinkmani clustered together with other Cryphodera spp. and with Meloidodera alni suggesting a monophyletic origin of non-cyst forming nematodes (Heteroderinae sensu Luc et al. ), which have been considered close to the ancestor of most species of Heteroderidae. Histological observations of P. parviflora feeder roots infected by C. brinkmani indicated that nematode females induce similar anatomical alterations to those reported for C. kalesari, consisting of formation of a single uninucleate giant cell (nurse cell) with hypertrophied nucleus, prominenet nucleolus, thickened cell wall and expanding into the stele and in contact of xylem, vacuum cambium and phloem. These findings are in agreement with the results of the phylogenetic analysis and indicate a close relationship in the plant responses induced by Cryphodera nematode females with those caused by the genetically related Meloidodera spp., which also induce formation of a uninucletate giant cell. [ABSTRACT FROM AUTHOR]