Elena Fanelli

Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region

Plant parasitic nematodes belonging to the genus Pratylenchus, also known as root lesion nematodes, cause serious economic damage to different crop plants. In order to explore genetic structures in different isolates, we investigated several P. thornei,P. neglectus and P. penetrans populations of different geographic origins. The analysis at the species level was also extended to P. penetrans, P. pinguicaudatus, P. vulnus and P. mediterraneus. Sequence analysis of a specific portion of DNA was carried out. In particular, the sequences of the D3 region of the 26S gene were obtained and compared with similar sequences available in databases. The results support the hypothesis that P. penetrans may represent a species complex, while in P. neglectus the intra-species heterogeneity observed is due to intra-individual variability. Furthermore, the specific conservation of some nucleotides in different P. thornei populations indicates their fixation in the rDNA repeats in this species. The presence of these nucleotides, the molecular signature of P. thornei, may assist in determining the nature of nematode infections.

Heterodera elachista the Japanese cyst nematode parasitizing corn in Northern Italy: integrative diagnosis and bionomics.

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.

Redescription and molecular characterisation of Xiphinema barense Lamberti et al., 1986 (Nematoda: Longidoridae) from wild olive trees in southern Italy

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.

Oscheius onirici sp. n. (Nematoda: Rhabditidae): a new entomopathogenic nematode from an Italian cave

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.

Morphological and molecular identification of potato and cereal cyst nematode isolates from Algeria and their phylogenetic relationships with other populations from distant theirgeographical areas

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.

Characterization of nematode resistance gene analogs in tetraploid wheat

Abstract Using a PCR-based approach we have isolated and characterized a putative plant resistance gene from Triticum turgidum whose corresponding resistance gene has been identified in Triticum aestivum . Furthermore, the partial sequences of four new resistance gene analogs (RGA), were used to study their expression profiles in leaves and in roots of two different varieties of T. turgidum . The analysis of the conserved motifs clearly indicates that the sequences we have isolated belong to the NBS-LRR class of plant resistance genes. Expression studies in healthy plants, using the varieties Creso and Latino of T. turgidum , indicate different localizations of RGA transcripts and suggest that they function in the general mechanism of plant surveillance against pathogens.

Detection and molecular characterization of the rice root-knot nematode Meloidogyne graminicola in Italy

The rice root-knot nematode Meloidogyne graminicola was detected in North Italy attacking roots of rice plants in lowland and upland fields. This nematode was previously reported only in South and Southeast Asia, China, South Africa, United States, Columbia and Brazil attacking rice and wheat. The present study provides an integrative diagnosis of the nematode on the basis of morphological and molecular data by sequencing the SCAR species specific marker, the ITS region and the partial mitochondrial coII-16srRNA genes. Morphological and morphometric traits of the new intercepted population of M. graminicola agree with the original description. The diagnostic SCAR marker was amplified showing the expected size fragment and unequivocally identified the Italian population as M. graminicola. Sequence analyses of ITS and coII-16SrRNA confirmed this identification. Sequence comparison of both molecular markers among Italian M. graminicola populations from lowland and upland rice fields revealed 100% identity. Phylogenetic analysis based on the ITS and coII-16SrRNA sequences revealed that Italian M. graminicola formed a well supported cluster with M. graminicola populations from GenBank confirming the high intraspecific variability among all populations. No correlation between genetic variability and geographic origins occurred. Furthermore, the phylogenetic trees suggest a common ancestor for most of the facultative meiotic parthenogenetic Meloidogyne species.

The morphological and molecular identity of Longidorus piceicola Lišková, Robbins & Brown, 1997 from Romania (Nematoda, Dorylaimida)

Abstract Longidorus piceicola, a new geographical and host record from Romania, was described and illustrated on the basis of two populations originating from a coniferous and a deciduous forest. The main morphological characters of specimens from Romania correspond very well with the type material collected from the soil around Picea abies L. (Slovakia) except for the shorter body and tail. The D2-D3 fragment of 28S rDNA from both populations was amplified and sequenced, and the sequences were identical to L. piceicola sequence from Slovakia. The partial 18S-ITS1-5.8S-ITS2 rDNA regions from one of the populations were sequenced for the first time. The evolutionary relationships between L. piceicola and the closest species L. intermedius based on D2-D3 sequence divergence and single-nucleotide polymorphisms are discussed. Although having very low sequence dissimilarity (0.3–0.9 %) both species have distinct morphology and biology. Longidorus piceicola differs from L. intermedius in having a much longer odontostyle, body, distance anterior end - guide ring, a wider lip region, more ventromedian supplements (11 vs 5–7) in the male, and develops through four rather than three juvenile stages. Furthermore, L. piceicola occurs more frequently in association with conifers, while L. intermedius is found mainly in oak forests.