High infection rates of wild olive (Olea europaea sp. sylvestris) feeder roots and soil infestation by a new root-knot nematode were found in sandy soil at Vejer de la Frontera (Cádiz), southern Spain. Morphometric traits and analyses of the nematode esterase electrophoretic pattern as well as of the internal transcribed spacer 1 (ITS1)-5.8S gene and D2-D3 fragment of the 28S gene of rDNA showed that specimens differed clearly from known root-knot nematodes. Studies of host-parasite relationships showed a typical susceptible reaction in naturally infected wild olive plants and in olive planting stocks (cvs. Arbequina and Picual) artificially inoculated with the nematode. However, the nematode did not reproduce in artificially inoculated chickpea, pea, and tomato. Because of the ability of this new nematode to infect wild and cultivated olives only, we suggest the common name, "Mediterranean olive root-knot nematode." The species is herein described and illustrated, and named as Meloidogyne baetica n. sp. The new root-knot nematode can be distinguished from other Meloidogyne spp. by (i) the perineal pattern, which is almost similar to that of M. artiellia, characterized by distinct inner striae forming two distinct longitudinal bands, extending throughout the perineum to just below the vulva; (ii) female excretory pore anterior to the level of stylet knobs, excretory pore distance from anterior end/length of stylet ratio extremely small (0.5 to 0.8); and (iii) second-stage juveniles with elongate-conoid tail. Phylogenetic trees derived from maximum parsimony analyses showed that M. baetica is closely related to M. artiellia, the cereal and legume root-knot nematode.

Thelazia callipaeda, commonly known as the 'oriental eyeworm', has been recently reported in Italy and other European countries. The insect/s that act as intermediate hosts and details of larval development inside the vector remain unclear. In order to (1) demonstrate the species of fly that may act as vector/s for T. callipaeda in southern Italy (Site A) and China (Site B) and (2) describe the larval development of the nematode in the body of flies, 847 Phortica (Drosophilidae) flies were collected from the above two sites, each with a history of human and/or canine thelaziosis. Flies were identified as Phortica variegata (245 - site A) and Phortica okadai (602 - site B), experimentally infected by 1st-stage larvae (L1), kept at different temperatures and dissected daily until day 180 post-infection (p.i.). Dead flies from site A were subjected to specific polymerase chain reaction (PCR) assay to detect T. callipaeda. To demonstrate the role of Phortica as vectors of T. callipaeda, 3rd-stage larvae (L3) recovered from the proboscis of flies were deposited onto the cornea of the eyes of dogs and rabbits. Following dissection, 3 (2.9%) of P. variegata in site A were found to be infected by L3 in the proboscis on days +14, +21 and +53 p.i., compared with 26 (18.4%) of Phortica flies recorded as being positive by PCR. Sequences from positive PCR products were 99% identical to sequences of the corresponding species available in GenBank (AY207464). At site B, 106 (17.6%) of 602 dissected P. okadai were found to be infected by T. callipaeda larvae (different stages) and in total 62 L3 were recovered from the proboscis of 34 (5.6%) flies. The shortest time in which L3 were found was at day +14, +17, +19, and +50 p.i. respectively, depending on the environmental temperatures. Of 30 flies overwintered for 6 months, 6 L3 were detected at day +180 p.i. in 3 flies (10%). The biology of larval development was reconstructed on the basis of the dissection of 602 P. okadai-infected flies and the morphology of larval stages in the insect body described. The present work provides evidence that P. variegata and P. okadai act as vectors for T. callipaeda in southern Europe and in China, respectively. The phenomenon of overwintering is described here for the first time for T. callipaeda and discussed. Finally, the relationship between T. callipaeda and its fly vector is considered in light of disease prophylaxis and to model its dissemination into habitats and environments favourable to Phortica flies.

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 Ditylenchus gigas 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' from Fabaceae in recent literature. Morphologically, the new species is characterized by a body size 1AE5-2 times longer than the 'normal race', stylet delicate (11AE5-13AE0 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 hsp90 gene 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.

A description of a new species of plant parasitic nematodes of the genus Rotylenchus from the family Hoplolaimidae is given and a recognition of Rotylenchus jaeni comb. n., previously known as subspecies R. magnus jaeni, as separate species is proposed. Rotylenchus montanus sp. n. is characterized by a hemispherical lip region with six rarely seven annuli, stylet 33–37 µm, female tail rounded, regularly annulated tip with 12–18 annuli and phasmid located 2–9 annuli anterior to anus. Rotylenchus montanus sp. n. is close to species of the monosexual group R. arsenjevi, R. corsicus, R. fragaricus, R. helicus, R. indorobustus and R. neorobustus, by a number of specific characteristics resulting from its specific matrix code: A5, B1, C1, D4, E2, F2, G3, H2, I2, J2, K2. Molecular characterization of R. montanus sp. n. and other Rotylenchus species are provided using D2–D3 expansion segments of 28S and the ITS1 of rRNA genes. The D2–D3 of 28S rRNA and the ITS1–rRNA sequences of R. montanus sp. n. differed in one nucleotide and in 16–20 nucleotides from those of an unidentified Rotylenchus species from Russia, respectively. Molecular analysis of populations of R. magnus and R. jaeni comb. n. demonstrated differences in the D2–D3 and the ITS1–rRNA sequences. These genetic differences together with some minor morphological characters support that both subspecies should be considered as two cryptic sibling species and warranted their elevation to species rank. The result of phylogenetic analysis of Hoplolaimidae for 45 sequences of the D2 and D3 expansion regions of 28S rRNA gene using Bayesian inference analysis under the complex model is presented. Phylogenetic tree of Rotylenchus species represents seven moderate to highly supported lineages. Grouping of Rotylenchus species within other hoplolaimids and analysis of phylogenetic relationships within the genus Rotylenchus using the ITS1 of rRNA gene sequences are also discussed.

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. Comparative analysis 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 the high nucleotide variability, sometimes, can preclude its 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.

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.

No abstract

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 5 week 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.