Specimens of nematode belonging to Oscheius genis was isolated through the Galleria bait method from soil collected in a karst cave of Tuscany (Central Italy). Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region and the 18S rRNA gene were amplified and sequenced. BLAST search at the NCBI discriminate this new taxon, similar to other Oscheius. This species belongs to Dolichura group. Cuticle is finely annulated, stoma is short and cheilorhabdion is simple, not well cuticularized. Female body is almost straight upon fixation, the reproductive system is didelphic and tail is short, conoid with pointed tip. Males are rare and similar to female in general morphology except for smaller size. Male body is straight when heat-killed, testis is single, ventral reflexed. Thei show peloderan bursa , tail short rounded and spicules slender and small. Ingective juveniles are slender withelongate tail and have stoma morphology similar to adult. The nematodes were cultured in Petri dishes on several substrates: Nutrient Agar, Escherichia coli, Botritis cinerea, meat baby food, without satisfactory results. Only Petri dishes method with G. mallonella larvae produced IJs, suggesting the entomopathogenicityof this new taxon.

Morphological and molecular analyses of a stem nematode causing a severe disease on infected sowthistle (Sonchus bulbosus) plants, involving the formation of gall-like structures on infected leaves and stems, have led to the description of a new species named Ditylenchus oncogenus n. sp. Morphologically, the new species is characterized by a medium to large body size (all adults more than 1 mm in length); a delicate stylet (9.0-11.0 ?m long) with minute, rounded knobs; a long post-vulval uterine sac (c. 65% of the vulva-anus distance); six incisures at the lateral fields and characteristic D. destructor-pattern of spicules (with pronounced ventral tumulus and anteriorly pointed, less sclerotized, cuticle parts present within the lamina). The results of molecular analysis of rRNA gene sequences, including the D2-D3 expansion regions of 28S rRNA, internal transcribed spacer (ITS) rRNA, partial 18S rRNA gene, the protein-coding mitochondrial gene, cytochrome oxidase c subunit I (COI), and the heat-shock protein 90 (hsp90) gene, support the new species status. The results of a host-suitability test indicated that the new species does not parasitize potato (Solanum tuberosum) tubers and broad bean (Vicia faba) seedlings. Histopathological observations on naturally infected sowthistle tissues revealed that D. oncogenus n. sp. causes floral stem neoplasia and midrib leaf gall formation on the type, and to date only known, host. The galls were characterized by extensive hyperplasia, where several necrotic cells in the neoplasic area were directly damaged by feeding of the nematode, whereas a number of adjacent cells showed typical cytological changes, such as granulated cytoplasm with hypertrophied nuclei and nucleoli.

In this study we investigated the morphometric and molecular characterization of a liver encapsulated third-stage larval population of Anisakis spp. infecting Merluccius merluccius and Lophius piscatorius caught in the Adriatic Sea waters (southern Italy). A polyphasic approach based on PCR-RFLP profiles of the ITS region, mitochondrial COI (cytochrome c oxidase subunit 1), sequencing and molecular phylogeny of ITS and mitochondrial COI was used to identify Anisakis larvae collected from fish samples. PCR-RFLP analysis showed three banding pattern corresponding to the peculiar pattern of A. pegreffii. Sequence data from ribosomal ITS and mitochondrial COI were analysed by Neighbour Joining, Minimum Evolution and Maximum Parsimony methods to evaluate the phylogenetic relationships among A. simplex sensu lato. The phylogenetic trees obtained for both ITS and COI revealed the existence of three distinct clades for A. simplex sensu sricto, A. simplex C and A. pegreffii and the sequences obtained in this study clearly clustered together with A. pegreffii sequences present in the database. Histopathological observations of anisakid nematode specimens detected on the liver surface of M. merluccius are illustrated. Encapsulated specimens of the L3 stage of the nematode were similar in size and morphometry to those found into the peritoneal cavity. Anisakis larvae encapsulated on the liver surface within dense and pearl coloured envelops caused host hepatic tissue necrosis, large cavities and oedematous liver spots to the host.

The wide range of feeding types and the ability to adapt to seasonal succession make nematodes significant indicators of ecological conditions of the soil in which they occur. Soil nematophauna is highly sensitive to any environmental damage and, therefore, the analysis of soil nematode community can be a useful diagnostic tool of soil health changes caused by polluting agents, among which also pesticides. Traditional morpho-taxonomic techniques for analysis of soil nematophauna have been flanked or substituted by more innovative and quicker molecular tools. Effectiveness of morpho-taxonomic and molecular techniques was comparatively evaluated through the analysis of soil nematode community from three selected relatively undisturbed and disturbed sites in Apulia region (Italy). Nematodes for both analyses were extracted from 100g sub-samples of composite soil samples collected at each site. Specimens for morphological analysis were fixed in a 2.5% formaldehyde solution and then identified at family and genus level under an optical microscope. The maturity and trophic diversity indices were determined. Total DNA was extracted from the nematode community of each soil subsample and PCR amplification was performed by using the small subunit (18s) of the ribosomal DNA gene, as diagnostic marker. The 18s rDNA was selected because of the large number of 18s sequences in GenBank, the existence of an 18s-based phylogenetic tree and the conserved nature of this gene to ensure complete phylogenetic coverage of the phylum. Sequence analysis through BLAST allowed to classify most of them at genus level and some of them at species level. Few sequences showed no similarity with those present in the database suggesting that they are new for the scientific community. The maturityand trophic diversity indices were also calculated for genera identified at molecular level. Results confirmed that nematodes are good indicators of soil health, as showing a different level of disturbance for each of the three sampled sites. Both morpho-taxonomic and molecular tecniques showed to be effective, though morpho-taxonomic is more time-consuming and skilfulness-requiring whereas a molecular analysis is largely more expensive.

Stunted growth of large patches of cabbage CV. Lupini, associated with severe soil infestations by the root-knot nematodeMeloidogynejauanica and the protist Plasmodiophora brassicae, the casual agent of clubroot disease, was observed in severalfields at Castellaneta, province of Taranto, in southern Italy. The host-parasite responses of cabbage roots to parasitism by the twosoil-borne pathogens was studied and compared. In roots infected by P. brassicae, the plasmodia were present in cortex and pericyclecells, causing hypertrophy and hyperplasia, and developed into resting spores within host tissues. Parasitism of M. jauanicawas characterized by the establishment of distinct permanent feeding sites with giant cells in the cortex, endodermis and vascularparenchyma, which limit water and nutrient translocation.

La tutela e la gestione del suolo richiedono grande attenzione, viste le importanti funzioni ambientali, economiche, sociali eculturali che il suolo stesso svolge. I nematodi del suolo possono costituire degli utili bioindicatori dello stato di salute di unsuolo, in quanto sono in grado di rispondere prontamente ai cambiamenti ambientali (stress e inquinamento) e pertantol"analisi della loro distribuzione e attività può essere usata per stabilire lo stato di conservazione di un suolo. Ilbiomonitoraggio della nematofauna può essere condotto utilizzando diversi indici ecologici strettamente correlatiall"arricchimento di materia organica ed ai gruppi trofici dei nematodi presenti nel terreno (Maturity Index e EnrichmentIndex). Nel corso del presente studio sono stati campionati tre diversi siti della regione Puglia, localizzati uno in una riservanaturale, il secondo in prossimità di una centrale elettrica a carbone e il terzo in prossimità di una discarica, e per ciascunodi essi è stata condotta l"analisi della nematofauna presente utilizzando sia approcci morfologici che molecolari. Il DNAtotale della nematofauna presente in ciascun campione è stato estratto e sottoposto ad amplificazione mediante PCR. Il geneper il 18S rRNA è stato utilizzato come marcatore molecolare in quanto in banca dati esistono molte sequenze del 18S, talida coprire l"intero phylum dei nematodi. Sono state determinate circa 100 sequenze per ciascun sito. L"analisi dellesequenze mediante BLAST ha permesso di classificare le sequenze ottenute a livello di genere, alcune delle quali sono stateidentificate anche a livello di specie. Gli indici ecologici sono stati calcolati sia per i dati morfologici che per i datimolecolari rivelando che i risultati dei due approcci sono sovrapponibili. I siti analizzati si sono rivelati più disturbati incorrispondenza della centrale elettrica e della discarica rispetto al sito nella riserva naturale. Questi risultati preliminaridimostrano che i nematodi sono sicuramente dei buoni indicatori dello stato di conservazione di un suolo.

Morphological and molecular analyses confirmed the presence of Pratylenchus hippeastri in regulatory samples collectedin commercial bromeliad operations from genera Guzmania, Neoregelia and Vriesea in central and south Florida, USA. Specimens ofP. hippeastri from bromeliads contained males which were not detected in the type population from amaryllis and are described hereinfor the first time. The rDNA sequences of these males matched those of P. hippeastri female type material. Pratylenchus hippeastri androot-lesion nematodes from several hosts in Florida were characterised at the morphological and molecular level, whereas other samplesfrom Russia and South Africa were characterised at the molecular level only. Sequence and phylogenetic analysis using the ITS rRNAgene of these root-lesion nematodes revealed the presence of eight putative new species (spH1-H8) closely related to P. hippeastri.Here we describe two Florida representatives of the amphimictic root-lesion nematodes from Bahia grass (N1) and maidencane (N2),previously characterised by Inserra et al. in 1996 and Duncan et al. in 1999, as two new species phylogenetically related to P. hippeastriand named P. floridensis n. sp. and P. parafloridensis n. sp., respectively. The small round or oval (rarely rectangular and occasionallyoblong) and enlarged spermatheca and the bluntly pointed or subacute tail with smooth and occasionally indented terminus separateP. floridensis n. sp. from P. parafloridensis n. sp., which has a quadrangular spermatheca and a subhemispherical or bluntly pointed tailwith generally smooth and rarely indented terminus. However, these characters may overlap in some specimens making a morphologicalseparation problematic without the use of molecular analysis. The close phylogenetic relationships shared by the species characterisedin this study indicate that they are representatives of a P. hippeastri species complex.

Amphimictic populations of root-lesion nematodes with numerous males and females having three lip annuli, a functional spermatheca and non-areolated lateral field occur on sword fern (Nephrolepis exaltata) in Florida. Identified for decades as Pratylenchus penetrans, they appeared to be a morphologically separated species on the basis of a longer stylet (17.8-18.3 ?m) than P. penetrans (15-17 ?m) and different lip pattern in en face view (rectangular vs dumb-bell in P. penetrans). Morphologically similar amphimictic root-lesion nematodes have also been detected on flax lily in Costa Rica. Subsequent morphological observations indicated that these amphimictic root-lesion nematodes from fern and flax lily are closely related to the parthenogenetic species P. bolivianus, which has areolated lateral fields. In spite of the reproductive and morphological dissimilarities between these populations, their separation into different species was not supported by the results of molecular analyses of their DNA sequences. The populations used in these analyses included those that are amphimictic from Florida and Costa Rica and others that are parthenogenetic from the type locality in Bolivia, and geographically distant localities in Chile, China, Colombia and Europe. Phylogenetic analyses of the ITS and D2-D3 expansion segments of the 28S rRNA gene indicated that they belong to the same species, P. bolivianus, which consists of two morphotypes, P. bolivianus (am) amphimictic and P. bolivianus (pm) parthenogenetic, herein described and illustrated. Contradictory results were obtained by the analyses using a portion of the hsp90 gene. The phylogenetic study, which included sequences of other root-lesion nematodes, a topotype and geographical distant populations of P. zeae, revealed that P. bolivianus and P. zeae formed highly supported clades in the majority consensus trees. PCR with species-specific primers for rapid diagnostics of P. bolivianus and P. zeae were developed and tested.

A new longidorid nematode, Longidorus asiaticusn. sp., is described and illustrated from a population extracted from soil associated with the movement of crape myrtle (Lagerstroemia indica) flowering bonsai trees imported from China into Italy. The new needle nematode is characterised by a small body size (2.74-3.52mm), a bluntly-rounded lip region, ca 12 ?m wide, continuous with body contour,amphidial fovea pocket-shape with posterior end rounded not bilobed, a moderately long and flexible odontostyle ca 85 ?m long, stylet ring located at ca 37 ?m from anterior end, vulva almost equatorial (48-54 %), tail short, about 2/3 of its width, dorsally convex-conoid, with rounded terminus, with a c' ratio ca 0.7, bearing two pairs of caudal pores and male absent. Integrative diagnosis was completed with molecular data obtained using D2-D3 expansion segments of 28S rDNA, ITS-rDNA, and partial 18S-rDNA. The phylogenetic relationships of this species with other Longidorus spp. using D2-D3 expansion segments,ITS and partial 18S-rDNA indicated that L. asiaticus n. sp. clustered together with L. hangzhouensis, Longidorus sp. JH-2014, and L. camelliae: all of them sharing a common Asiatic geographic origin.

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 morphometricaltraits 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 bea 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 ofthe 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.

Morphologial, biochemical, molecular and karyological analyses of different populations and races of the stem and bulb nematode Ditylenchus dipsaci have suggested that it represents a species complex, of which only D. dipsaci sensu stricto and its morphologically larger variant, known as the giant race of the stem and bulb nematode, are plant parasites of economic importance. The present study singles out the giant race from this complex, herein described as a new species named Ditylenchusgigas n. sp., on the basis of morphological and molecular data obtained from several populations collected from broad beans in southern Italy, southern Spain and Lebanon. The new species epithet, which refers to the large body size of the nematode with respect to the normal races, must be considered to be conspecific with the D. dipsaci ‘giant race’ fromFabaceae in recent literature. Morphologically, the new species is characterized by a body size 1Æ5–2 times longer than the ‘normal race’, stylet delicate (11Æ5–13Æ0 lm long) with knobs distinctly sloping backwards, and long post-vulval uterine sac (81–150 lm long). Results of molecular analysis of rDNA sequences including the ITS1-5.8S-ITS2 region, the D2–D3 fragment of the 28S gene, the small 18S subunit, the partial mitochondrial gene for cytochrome c oxidase I (mtCOI), and hsp90gene sequences, support the new taxonomic species status for the former D. dipsaci giant race from Vicia faba, and clearly distinguish D. gigas n. sp. from D. dipsaci sensu stricto.

Hemicaloosia vagisclera n. sp. is described from Bermuda grass (Cynodon dactylon) in Florida. This new species is characterised by females with body slightly ventrally arcuate in death, face with a prominent elliptical oral disc, large amphids, slender stylet with mean length 64 ?m, lateral field consisting of a single longitudinal line marked by continuous and discontinuous transverse striae, oval and full spermatheca, sclerotised vagina vera and tail annuli width greater than that of remaining body annuli. Diagnostic characters for the males are a C-shaped body, head with 4-5 faint lip annuli and pronounced oval oral disc, lateral field with two longitudinal lines intersected by transverse striae, tail digitate in the distal portion posterior to bursa, distinctly annulated and with a round terminus. Molecular characterisation of H. vagisclera n. sp. using the D2-D3 domain of 28S rRNA, partial 18S rRNA and ITS rRNA gene sequences is also provided. The phylogenetic relationships of this species with other representatives of the suborder Criconematina are presented and indicated that H. vagisclera n. sp. has sister relationships with Caloosia longicaudata supportingthe classification of Caloosia together with Hemicaloosia as separate genera in the family Caloosiidae. A diagnostic PCR-ITS-RFLP profile for H. vagisclera n. sp. is also given together with an identification key for 7 known species of Hemicaloosia.

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.

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 suggestinga monophyletic origin of non-cyst forming nematodes (Heteroderinae sensu Luc et al. 1978), 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.

Root-lesion nematodes of the genus Pratylenchus are migratory endoparasites that are distributed worldwide and are regarded as severe constraints of many crops. Proper identification of these nematodes (> 70 species) is critical for their management and phytosanitary purposes. The morphological separation of Pratylenchus species is complicated by their high morphological plasticity, the small number of the differential characters available and high intraspecific variability of some of these characters. During the last decades, new approaches based on biochemical, molecular and phylogenetic analyses have provided powerful tools to nematode systematics and reliable identification of Pratylenchus spp. In particular the ITS, 18S and 28S ribosomal regions have been largely used to discriminate among different populations and species of Pratylenchus. The need for applying a polyphasic approach to characterize Pratylenchus species, and, in addition, to demonstrate the existence of cryptic or complex species, which are morphologically indistinguishable but genetically divergent, has been shown in recent morphological and molecular studies of members of this genus. The findings of these studies, conducted in the last decade, have produced a great number of new sequences and taxa used for testing hypotheses of phylogenetic relationships within Pratylenchus and have emphasized the importance of polyphasic taxonomy as a better strategy to delimit species and to infer phylogenetic relationships in this complex group of nematodes.

Root-lesion nematodes of the genus Pratylenchus have a worldwide distribution and are regarded as severe production constraints for numerous important crops. Correct and accurate identification of species within this important genus of plant-parasitic nematodes is critical but is complicated by their stenomorphic status. A polyphasic approach to diagnosis is therefore proposed as a better strategy for distinguishing species and to infer phylogenetic relationships within the genus. Recent studies on Pratylenchus spp. recovered from damaged plantain (Musa spp., genome AAB) in Ghana, West Africa, has resulted in their description asa new species, Pratylenchus speijeri n. sp. Morphological and molecular features of this species were compared with those of P. coffeae, its most closely related species, and other amphimictic species of Pratylenchus that have and undivided face with two lip annuli. Morphological characterisation of P. speijeri n. sp. did not result in an unambiguous separation from P. coffeae, whereas molecular analysis clearly distinguished the two species. Only a few and often variable morphological features, such as larger stylet knobs and a more frequently indented tail terminus separated these populations from P. coffeae. Sequences and phylogenetic analyses of D2--D3 of 28S rDNA and ITS containing regions of 60 individual nematodes from the P. coffeae species complex, sourced from various geographical locations, generated majority consensus BI trees with three major clades, where P. speijeri n. sp. formed its own separate clade from P. coffeae. During recent years the use of SEM, LM and molecular approaches to study P. coffeae, or representatives of the P. coffeae species complex, has resulted in the descriptions of several new cryptic species, namely P. jaehni, P. floridensis, P. parafloridensis and now P. speijeri n. sp. The morphological and molecular features of these cryptic species are compared and the practical application of such moleculardata discussed.

Nematodes are widely recognised as bioindicators of the soil environment health. Analysis of soil nematode community is increasingly used to calculate various ecological indices related to enrichment and trophic status of nematofauna. The soil nematode community from three selected relatively undisturbed and disturbed sites in the Apulia region (Italy) was comparatively studied through both morpho-taxonomic and molecular analysis. Nematodes for both analyses were extracted from 100 g sub-samples from composite soil samples collected at each site. Nematodes were fixed in a 2.5% formaldehyde solution and thenidentified at family and genus level under an optical microscope. The maturity and trophic diversity indices were determined. For the molecular study, total DNA was extracted from the nematode community of each soil subsample and PCR amplification was performed by using the small subunit (18S) rDNA, as diagnostic marker, for nematode species discrimination. The 300 sequences available at this moment are still under characterisation. Sequencing of further 18S amplicons is also in progress.

Soil nematodes are organisms that quickly respond to changes (stress and pollutants) in the environment and can be useful ecological indicators of environmental disruption. Since they occur in any environment containing organic carbon, they do not quickly escape from stressful conditions, occupy key positions in soil food webs and can be classified in easily identifiable trophic groups (Bongers and Ferris, 1999). While there are many indices of biological diversity, specific tools have been developed for nematodes, such as the Maturity Index (MI) and the 3 Enrichment lndex (El). These indices are based on an ecological classification where to each taxonomic family is assigned an ecological value that ranges from l (typical families of polluted soils or sediments) to 5 (typical families of soils or sediments). The lower values (1 and 2) belong to colonizer nematodes (c), i.e. opportunists, characterized by a rapid biological cycle and able to quickly invade unstable or polluted habitats. High values (3 to 5) belong to persister nematodes (p), characterized by a slow reproduction rate. Persisters are more sensitive to pollutants and 1 other disturbances than colonizers, therefore MI also serve to measure the impact of mixtures of pollutants and the effect of their complex interactions with biotic and abiotic environment. The aim of this study was to identify the nematofauna, recovered from three different habitats, at morphological and molecular level to provide useful information on the soil features and any possible disturbances by calculating ecological indexes of soil biodiversity

Helicotylenchus is a cosmopolitan genus including some important plant parasiticnematodes causing damage in agricultural and horticultural crops. During a soil sampling ofalmond (Prunus) orchards in Baft region of Kerman province (South East of Iran), a species ofHelicotylenchus was extracted from rizosphere of almonds and identified according tomorphological and morphometric characters as H. digonicus. In addition, the D2D3 segment of 28srDNA was amplified using specific primers and sequenced. The Nblast result showed that studiedpopulation has 8 nucleotides differences with Italian population of H. digonicus (DQ328758; 99%identity). Phylogenetic analysis using Maximum Likelihood places this population close to anundescribed population of Helicotylenchus (HM014303) and H. digonicus. Genetic pairwisedistances showed low variation (0.002) with a population of H. digonicus (HM014241) from SouthAfrica.

The occurrence of a male-less population of Sphaeronema alni parasitising chestnut (Castanea sativa) roots and inducinga stelar syncytium is reported for the first time in Pola de Somiedo (Oviedo province), Spain.Morphometric and molecular characters ofthe Spanish population matched those of a topotype population from Russia. SEM observations showed swollen females having the firstlip annulus wider than the second and appearing as a cap-like, circumoral elevation. The second-stage juveniles, having a single band inthe lateral fields, were characterised by a non-annulated dome-shaped lip region derived from the fusion of the oral disc with all the lipsectors and lip annuli, and showing slit-like amphidial apertures and an oval prestoma. The sequences of the D2-D3 expansion segmentsof 28S rRNA, partial 18S rRNA and ITS rRNA gene for the Spanish and topotype populations of S. alni were congruent and matchedthose deposited in GenBank for another population from Germany, thereby confirming their conspecificity. A PCR-RFLP profile ofD2-D3 of 28S rRNA for identification of this species was also provided. The phylogenetic relationships between S. alni populationsand representatives of the suborder Criconematina, as inferred from analysis of partial 18S rRNA and D2-D3 of 28S gene sequencesobtained in this and previous studies, indicated that S. alni formed a basal clade on the majority consensus Bayesian phylogenetic trees,standing together with Meloidoderita sp. or alone. These findings provide additional evidence of the need to clarify the position ofSphaeronema within Criconematina and its relationships with representatives of Tylenchulinae.

This study reports on the isolation and characterization of four different endoglucanases in the root-lesion nematode Pratylenchus vulnus. The gene structures of two of these, Pv-eng-1 and Pv-eng-2, were fully determined, and Pv-eng-3 and Pv-eng-5 were partially sequenced. Spatial expression of Pv-eng-1, Pv-eng-2 and Pv-eng-5 was examined by in situ hybridization. Pv-eng-1 and Pv-eng-2 transcripts were localized in the subventral oesophageal glands, whilst the Pv-eng-5 transcript was localized in the intestine. Real-time RT-PCR showed that three of the four endoglucanases had the highest transcriptional level in adult males and females, thus demonstrating that adults are also parasitic in P.vulnus. Mixed stages of P.vulnus were treated with double-stranded RNA (dsRNA) of Pv-eng-1 in order to study the effect of gene silencing (RNAi). Silencing Pv-eng-1 by dsRNA targeting of the carbohydrate-binding module (CBM) resulted in a significant reduction (88-98%) of the transcript level, suggesting that P.vulnus is susceptible to RNAi. Furthermore, silencing P.vulnus showed a reduction (54%) in nematode reproduction on carrot minidiscs over a 5week period. These results suggest that silencing of Pv-eng-1 may result in reduction of the ability of the nematode to locate and invade roots and, therefore, to establish and reproduce.

Sequence comparisons and molecular phylogenetic analyses were used to describe the nucleotide variability of the ITS containing regions of eighteen Pratylenchus species and several populations. Comparativeanalysis of nucleotide sequences of the rDNA internal transcribed spacers (ITS1 and ITS2) among Pratylenchus species used in the present study demonstrates that ITS sequences can widely vary in primary sequence and length. Alignment of eightyseven Pratylenchus sequences and one outgroup taxon reveals the presence of ambiguous regions that have the greatest effect on phylogeny reconstruction. Phylogenetic analyses using Bayesian Inference, Neighbour Joining-LogDet, Maximum Likelihood and Maximum Parsimony, distinguished twelve highly or moderately supported major clades within Pratylenchus. Our results support the taxonomic usefulness of the ITS region to identify root-lesion nematode species of the genus Pratylenchus but thehigh nucleotide variability, sometimes, can precludeits use to resolve relationships among all members of the genus. In addition, the phylogenetic groupings are not congruent with those defined by characters derived by lip patterns and numbers of lip annuli.

Root-lesion nematodes of the genus Pratylenchus are migratory endoparasites distributed worldwide and regarded as severe constraints of economic crops. Crop damage is often aggravated by their interactions with soilborne fungi and bacteria, resulting in complex diseases which are biological and physiological rather than physical in nature. Morphological identification and species delimitation of these nematodes is still problematic due to their high morphological plasticity, the small number of diagnostic features available at species level, the intraspecific variability of some of these characters and many incomplete descriptions published in the literature. Thus, molecular approach, by using the ribosomal DNA, plays a key role within the taxonomy of this genus. The ITS containing region of eighteen species of Pratylenchus was sequenced and the variation within and between species was assessed. Phylogenetic analyses were conducted by using different tree reconstruction approaches: Bayesian inference (BI), neighbour-joining Log-Det (NJ), maximum parsimony (MP) and maximum likelihood (ML). The alignment revealed small species-specific DNA sequences suitable for the construction of potentially useful species- specific primers or for a more promising approach for DNA barcoding of root-lesion nematodes.

Molecular characterisation of four Schistonchus caprifici populations (two from Spain and two from Italy), using the partial18S, the D2-D3 expansion segments of 28S of ribosomal DNA (rDNA) and the partial mitochondrial gene for cytochrome c oxidaseI (mtCOI), is provided. Amplicons from partial mtCOI were analysed using ‘Single Strand Conformation Polymorphism’ (SSCP)analysis in order to rapidly screen for genetic (haplotypic) variability. SSCP analysis of mtCOI revealed a close relationship betweenthe Spanish and Italian populations with low intra-population variability and only two haplotypes were detected.Mitochondrial COI andribosomal genes were analysed using Maximum Likelihood and Bayesian inference to reconstruct phylogenetic relationships withinS. caprifici and other members of the family Aphelenchoididae. The phylogenetic analysis based on mtCOI showed no geographicvariability among Italian, Spanish and Turkish populations. The results of phylogenetic analyses based on the D2-D3 expansionsegments of 28S rDNA region and the partial 18S rDNA genes revealed a closer phylogenetic relationship between S. aureus,S. laevigatus, S. virens and S. centerae, whilst S. caprifici and S. guangzhouensis clustered separately. The phylogeny of the genusSchistonchus was well related to some morphological characters, such as position of excretory pore, presence/absence of labial discand number and position of caudal papillae. The monophyly of the genus Schistonchus was rejected by the Shimodaira-Hasegawa testbased on tree topologies

Several juvenile and adult nematodes were isolated after dissection of pupae and adults of the red palm weevil, Rhynchophorus ferrugineus, recovered from an infested Phoenix canariensis Chabaud exemplar in Bari, Italy. Two species of nematodes were recovered, Teratorhabditis synpapillata and Mononchoides macrospiculum n. sp. which is described herein. The mitochondrial cytochrome oxidase I (COI), the ITS-containing region, the 18S rRNA gene (SSU) and the D2-D3 expansion domains of the 28S rRNA gene (LSU) were amplified and sequenced. The new species, M. macrospiculum n. sp., is described at morphological and molecular level. Phylogenetic analyses using SSU and LSU sequences placed M. macrospiculum n. sp. together with M. composticola and M. striatus. The sequences of the Italian population of T. synpapillata are identical to those of T. synpapillata from Japan. This is the first report on the association of M. macrospiculum n. sp. and T. synpapillata with the red palm weevil in Europe.

A new strain of Steinernema carpocapsae (Weiser, 1955) was isolated from soil collected in a lagoon plain in Veneto region (North-East Italy). This new strain was named ItS-CAO1. Molecular and morphological analyses were performed. The ITS region and the 18S rRNA gene were amplified and sequenced. The ITS products were then digested with six restriction enzymes in order to unequivocally identify this species. Nematode virulence was tested against last instar of Galleria mellonella (L.) using different laboratory assays. Insect mortality of this new strain is very high in penetration (100%) and sand column assay (93.3%) and the percentage of penetrating infective juveniles was 57.6 and 42.9, respectively. Larval mortality in one-on-one quality assay was 50% and in exposure time assay it was 50% at 19 minutes. With the results of infectivity assays we can evaluate the possibility to use this new strain in biological control programs.

A population of Xiphinema macroacanthum Lamberti, Roca & Agostinelli, 1989 originating from olive orchards in Brindisi, Italy and containing both adults and all juvenile stages, is described and illustrated. The first juvenile stage is reported for the first time. Molecular characterisation of this species, using the D2-D3 expansion domains of the 28S rDNA and ITS region, was carried out. PCR-RFLP analyses of the ribosomal gene regions determined species-specific patterns that clearly differentiate X. macroacanthum. Sequences of the D2-D3 domains and the partial 18S-ITS1 rRNA genes were analysed using several methods for inferring phylogeny to reconstruct the relationships between X. macroacanthum and other Xiphinema species.

A nematode survey conducted in 2013 in Algeria, revealed that potato cyst nematodes (PCN) and cereal cyst nematodes (CCN) are widely distributed in several potato and cereal growing regions of the country. Sixteen PCN populations from five localities and five CCN populations from four of these localities were collected and characterized at the morphological and molecular levels. The PCN populations were identified as Globodera rostochiensis and G. pallida occurring separately or in mixed populations. Two species of CCN were detected. Heterodera avenae was found in four localities, whereas H. hordecalis only in one locality in association with H. avenae. The morphological and morphometric identification of PCN and CCN was confirmed by diagnostic ITS-RFLP profiles and sequencing. Phylogenetic analysis of the ITS, D2-D3 expansion domains of the 28S rRNA gene and 18S rRNA gene was made for PCN and CCN populations. Globodera pallida and G. rostochiensis from Algeria show great similarity with European and South American populations. Because of the high divergence among Algerian populations of G. pallida and G. rostochiensis it can be assumed that they were multi-introduced in Algeria. The most divergent population of G. pallida, that formed a well-separated group with some populations from Chile and Peru, suggests a later or independent introduction of this population into Algeria. Heterodera avenae and H. hordecalis formed a well-supported cluster with the corresponding populations.

Several nemamdes, juveniles and adults, were found dissecting some Rhynchophorus ferrugineus pupae and adults from infested Phoenix canariensis exemplar in Bari (Italy). Insect was intact externally but inner tissues were completely liquefied. Nematodes were collected using the water trap method and total DNA was extracted from each individual. The 18s rDNA, the ITS containing region and the mitochondrial cytochrome oxidase I (COI) were amplified md sequenced. ITS-RFLP analysis were also obtained. BLAST search revealed that nucleotides sequences are similar (93%) to Koerneria sp. R81982 (Nematoda: Diplogastidae). Nematodes belonging to Diplogastridae are commonly associated with kts, with different types of association depending on diplogastrid genera. Koerneria spp. are frequently associated with stag and dung hties. Characterization studies are now still in progress for the species identification. Our future purpose is to clarify the kind of association between this specie and the Red Weevil and the eventual role as natural control agent.

Vengono qui illustrati i risultati delle osservazioni effettuate in Italia meridiona- le sulla biologia del nematode fitoparassita Schistonchus caprifici Cobb (Nematoda, Aphelenchoididae).?Il nematode, inserito nel sistema mutualistico "Nematode-Blastophaga-Ficus", si riproduce sia sul fico selvatico Ficus carica L. var. sylvestris Auct. sia sul fico commestibile F. carica Linnaeus ed è veicolato dall'imenottero impollinatore Blastophaga psenes (Linnaeus) (Agaonidae), la cui riproduzione però avviene esclusivamente sul fico selvatico. Mediante osservazioni al microscopio di se- zioni istologiche di tessuti floreali e dell'addome di esemplari di B. psenes, sono stati evidenziati gli effetti del fitoparassitismo del nematode sull'infruttescenza del fico e la stretta relazione di trasporto passivo tra imenottero impollinatore e nematode. Il nematode risulta parassita dei fiori femminili del fico con densità di popolazioni che variano da 0 a 12.000 esemplari per grammo di tessuti floreali. Durante la sua attività trofica esso induce necrosi ed ipertrofia cellulare nei tessuti epidermici e parenchimatici, con evidenti ispessimenti delle pareti cellulari dei tessuti floreali interessati dal parassitismo, mentre numerosi esemplari del nematode si aggregano nell'addome della vespa vettrice per essere poi trasportati e disseminati durante l'ovideposizione.In conformità alle altre specie di Agaonidae, B. psenes è caratterizzata da un marcato dimorfismo sessuale con femmine dotate di ali, mentre il maschio è attero.?Conseguentemente il nematode, per ovvie necessità di trasporto, è presente so- lamente nell'addome delle femmine alate che volando raggiungono i ricettacoli nella fioritura successiva del caprifico per la deposizione delle loro uova. Durante questa fase di riproduzione dell'impollinatore si compie la dispersione del fitoelminta.

Oscheius onirici sp. N. (Nematoda: Rhabditidae) was isolated from a karst cave soil of Central Italy. Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region, the D2-D3 expansion domains of the 28S rRNA gene and the 18S rRNA gene were amplified and sequenced. BLAST search at NCBI by using all molecular markers revealed that this taxon is similar to Oscheius species. Phylogenetic trees of ITS, 28S and 18S rDNA revealed that O. onirici sp. N. belongs to Dolichura-group. Oscheius onirici sp. N. is characterized by small body size and stoma rhabditoid type. Female reproductive system is amphidelphic. Males are rare with peloderan bursa, spicules slender and small, nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern. Entomopathogenicity bioassay revealed that this nematode is capable of infecting larvae of Galleria mellonella and Tenebrio molitor.

Endo-1,4-?-glucanases have been found in numerous plant-parasitic nematodes (PPN) and play key roles in nematode--plant interactions. Four ?-1,4-endoglucanase encoding transcripts were cloned and characterised in the root-lesion nematode Pratylenchus vulnus. The P. vulnus endoglucanases show high similarity to other endoglucanases found in other nematodes belonging to glycosyl hydrolase family 5 (GHF5). All deduced proteins from the cloned sequences contained the predicted signal peptide for secretion and three of the four endoglucanases did not contain a carbohydrate-binding module (CBM). Real-time PCR experiments suggested that two of endoglucanase transcripts are expressed through the second-stage juveniles (J2), J3--J4 juveniles, males, and the adult females at different amounts confirming that all life stages are able to penetrate the host plant. In-situ hybridisation showed that both transcripts of endo-1,4-?-glucanases accumulated specifically in the pharyngeal subventral gland cells of all P. vulnus stages, thus suggesting the parasitic behaviour of each life stage. Recent data on these characterised genes will be presented and discussed.

PESTOLIVE (Contribution of olive history for the management of soil-borne parasites in the Mediterranean basin) is a project funded by ARIMNet, an ERANET action supported by the 7th European Framework Programme and by Mediterranean non-European countries.PESTOLIVE aims at producing knowledge and tools for new and efficient management of plant-parasitic nematodes (PPN) and plant-pathogenic fungi (PPF) in olive (Olea europaea L.) cropping systems and nurseries, while reducing the use of pesticides. Because of the anthropic continuum from Olea post-glacial refuges to oleasters (domestication) and then to olive-trees (breeding and cropping), the fragmentation of the PPN and PPF communities and of their natural enemies could explain the scattered diversity of the control techniques (especially resistant rootstocks, biocontrol, cropping strategies) developed and applied all around the Mediterranean basin. The novelty of PESTOLIVE is based on i) the analysis and the management of the parasite diversity (ecology of communities) instead of controlling emblematic species (population approach) and ii) the involvement of knowledge about the historical co-adaptation of soil-borne parasite and natural enemies communities to olive-tree domestication (origins and past assemblages) and breeding that follows the history of O. europaea around the Mediterranean basin.

Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. The current study focuses on the unexplored coastal zone of Lebanon where soil samples were taken in different sites chosen randomly along the coast like beaches, agricultural and herbaceous fields. In total, 350 soil samples were collected, mainly from the southern part of the country. An integrated approach, combining both traditional (morphological) and molecular methods, was used to characterize entomopathogenic nematode species encountered. Two named-species are added to the EPNs catalog in Lebanon from 4 samples out of the total 350 samples isolated: Heterorhabditis indica, reported for the first time in the country (samples AYAB6 and BRA20) and Steinernema feltiae (samples ANFA5 and EDA1). Furthermore, one undescribed potential entomopathogenic nematode belonging to Oscheius genus was recovered. The symbiotic bacteria from S. feltiae and H. indica were also molecularly identified through the use of five gene fragments recA, gyrB, dnaN, gltX and infB. Phylogenetic relationships of entomopathogenic nematodes and their symbiotic bacteria were inferred by using maximum-likelihood analysis. Soil studies were subsequently carried out in order to assess a possible relationship between soil parameters and their effects on EPNs. Results indicate that sandy texture and moisture are key factors for the presence and survival of EPNs in the soil in Lebanon.

A new root-lesion nematode, particularly pathogenic to Musa spp. and causing important plantain losses in Ghana, is described and named Pratylenchus speijeri n. sp. The cryptic status of this species within the P. coffeae species complex has been assessed and confirmed in this study. An extensive comparison of the morphological and molecular characteristics of this new species with those of P. coffeae and other related amphimictic species did not result in an unambiguous separation of this species from P. coffeae because only a few morphological features of diagnostic value were found. Sequence and phylogenetic analyses of the D2-D3 expansion segments of the 28S rRNA gene, the ITS rRNA gene and a portion of the hsp90 gene of P. speijeri n. sp. and P. coffeae species complex populations from different sources generated majority consensus BI trees with three major clades: P. speijeri n. sp. from Musa spp. roots in Ghana; unidentified or putative new Pratylenchus sp. C1 from Colocasia esculenta roots in Japan and P. coffeae with non-homogeneous relationships from different hosts and distant geographical areas. These results confirmed the validity of P. speijeri n. sp. as a new taxon and indicated that P. coffeae populations from Colocasia in Japan also need to be considered as a new species. Sequence differences in the ITS were used to design group- and species-specific primers to detect P. speijeri n. sp. and other species of P. coffeae species complex. The use of these species-specific primers for the separation of P. speijeri n. sp., Pratylenchus sp. C1 and P. coffeae has important practical application in breeding programmes for agriculture in West Africa. © Koninklijke Brill NV, Leiden, 2012.

A population of Xiphinema barense from wild olive trees in Torre Pozzella, Brindisi province, southern Italy, is described using both morphological and molecular studies and compared with the description of the type specimens. The wild olive nematode population agrees very well with all morphometrics provided in the original description. However, detailed observations of the lumen of the tubular portion of the uterus in paratypes and specimens of the new population revealed a clear pseudo-Z-organ with small granules mixed with crystalloid bodies which were previously undetected. Photomicrographs of adult paratypes, which were lacking in the original description, and of specimens of the new population from wild olive trees are provided. The results of the phylogenetic analyses based on the sequences of the D2-D3 expansion regions of the 28S rRNA gene and ITS rRNA genes confirm the species differentiation and indicate the phylogenetic position of X. barense and its relationship with closely related species.