Franco Lamberti

A molecular phylogenetic approach to Longidoridae (Nematoda: Dorylaimida)

The Longidoridae are a group of ectoparasitic nematodes including two subfamilies and six genera with hundreds of species. Sequences of the D2 and D3 expansion region of the large subunit (LSU) rRNA nuclear gene were amplified and used to reconstruct the phylogeny of longidorids. Phylogenetic analyses with maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) were performed with one outgroup taxon and 62 longidorid sequences. Confidence of inferred clades was assessed by non-parametric bootstrapping for MP and Bayesian posterior probability for ML. All analyses placed Paralongidorus species as an inner group within the otherwise monophyletic genus Longidorus. The genus Xiphinema, except for X. americanum-group species, was placed as the sister group of Longidorus with strong support from the ML and BI analyses. The X. americanum-group was strongly supported as an exclusive clade to other genus Xiphinema species. The position of the Xiphidorus clade was not well resolved and the phylogenetic analyses did not support it as a sister group to Longidorus as previously inferred from morphology. Secondary structure models were constructed for the D2/D3 region of LSU rRNA for all studied species. It was found that sequence-based and structural morphometric rRNA phylogenies were incongruent.

Molecular and morphological consilience in the characterisation and delimitation of five nematode species from Florida belonging to the Xiphinema americanum -group

Taxonomic keys and original descriptions were used to identify 26 Xiphinema americanum-group populations from Florida comprising X. georgianum (eight populations), X. citricolum (six), X. floridae (six), X. laevistriatum (five) and X. tarjanense (one). Principal component analysis of a subset of 19 morphometric characters accorded with the species designations; discriminant analysis of six characters assigned 93% (111 of 119) of the specimens to the correct putative species. A phylogeny of these populations estimated from analyses of rDNA sequences (ITS and D2D3) was also congruent with species designations from taxonomic keys and PCA. The D2D3 sequences revealed very little intraspecific variation whereas each population sampled produced a unique ITS sequence. Intraspecific variation in the suites of character code values from polytomous keys resulted mainly from minor discrepancies between population character means and reported character ranges for the species. We show that, for these taxa, species delimitation based on the requirement that sister taxa evolve autapomorphies distinguishes intraspecific variation from phylogeny and, as applied to molecular characters, delimits the same taxa as those predicted by morphological keys and PCA.

Isolation and characterisation of microsatellites for Xiphinema index using degenerate oligonucleotide primed PCR

A short insert genomic library for Xiphinema index, the natural vector of Grapevine Fanleaf Virus, was constructed from degenerate oligonucleotide primed PCR (DOP-PCR) products. The genomic library was screened for (CA)n microsatellites. Screening of 6200 colonies and comparison of the sequencing results revealed seven (CA)n containing microsatellites, coded here as XIMSL1, XIMSL2, XIMSL3, XIMSL4, XIMSL5, XIMSL6 and XIMSS1. XIMSL prefixed microsatellites were followed by the motif of the same long interspersed element. Microsatellite XIMSS1 has some similarity to the short interspersed element. Except for XIMSL1, all microsatellites were proven to be effective diagnostic tools at species level. Genetic diversity between and within populations was also evaluated for each microsatellite.

Identification and histopathology of the foliar nematode Aphelenchoides ritzemabosi (Nematoda: Aphelenchoididae) on basil in Italy

The foliar nematode species Aphelenchoides ritzemabosi is reported attacking sweet basil in glasshouses in the province of Imperia, northwest Italy. The nematode colonised and reproduced within the leaf, petiole and stem tissues. The epidermis and mesophyll were invaded by the nematode which caused internerval discoloration and necrosis and collapse of the palisade and spongy parenchyma. All nematode developmental stages, including eggs, were observed in the leaf tissues. Morphology and morphometrics of A. ritzemabosi from chlorotic and necrotic leaf tissues are presented. Possible control measures, which are complicated by the short life cycle of the nematode, the broad host range, and the short productive cycle of basil, are also discussed, together with the risk of an erroneous field diagnosis caused by difficulty in differentiating the symptoms of basil downy mildew and basil black spot from those of the foliar nematode.

Morphological and molecular characterisation of Longidorus danuvii sp. n. and L. silvae Roca, 1993 (Nematoda: Dorylaimida) from Serbia

Specimens of a Longidorus species, here described as L. danuvii sp. n., were recovered from the rhizosphere of Amorpha fruticosa growing in a floodplain forest on the bank of the river Danube at Novi Sad, Serbia; a second population was collected from Populus sp. also at Novi Sad. Longidorus danuvii sp. n. is characterised by medium sized body length (3.7-5.4 mm), slightly expanded, anteriorly flattened and laterally rounded lip region, set off by a depression from the rest of the body, distinctly bilobed amphidial pouches with lobes of about equal length, medium odontostyle length (86-101 μ m) and elongate, conoid tail with more or less bent appearance. Four juvenile developmental stages were identified. A second Longidorus species, identified as L. silvae, was recovered from the rhizosphere of Quercus petraea at Rakovac, Fruska Gora mountain, Serbia. The discovery of two males is the second record for this species. PCR-RFLP analyses of the ITS containing regions and sequences of the D3 region of the 26S rDNA were done.

Cuticlin Gene Organization in Meloidogyne Artiellia

The cuticlins are insoluble, non-collagenous components of the nematode cuticle. They were first described in Ascaris lumbricoides by Fujimoto and Kanaya in 1973. Their insolubility, even in the presence of strong detergents and reducing agents, indicates that they are highly cross-linked by non reducible bonds. Recently, Sebastiano et al. (1991) have identified and cloned two distinct cuticlin genes from Caenorhabditis elegans, namely cut-1 and cut-2.

SOD polymorphism in the Xiphinema americanum -group (Nematoda: Longidoridae)

Isoelectrofocusing of superoxide dismutase (SOD) isoforms was carried out on the extracts of 117 nematode populations belonging to the so-called Xiphinema americanum -group. These populations came from the USA (77), Chile (5), Argentina (1), Venezuela (5), Portugal (15), Italy (2), Crete (1), Montenegro (1), Slovakia (4), Hungary (3), Egypt (1) and India (2). A total of 17 bands of enzyme activity were observed in the screening, whilst single enzyme phenotypes showed from two to eight bands. The high degree of SOD polymorphism of this nematode collection was grouped by cluster analysis into seven distinct homogeneous groups characterised by specific combinations of SOD markers. Sub-groups could be discriminated for larger groups. The small Groups 3 and 5 were constituted mostly by populations from USA east coast states ( i.e ., NY and PA, respectively). The larger Group 1 resulted from the association of populations coming from various and distant North American States. In other large groups, North American populations were associated with South American and European populations. Overall, the data presented here suggest that geographic separation and different hosts do not seem to be the source of genetic diversity for the X. americanum -group. When an adequate number of populations were collected from the same country, the variability expressed by such a sub-sample was comparable to that of the whole nematode collection. For the first time, homogeneous populations of a large collection of X. americanum -group populations were associated by molecular means in order to explore further approaches that may help resolve the recalcitrant taxonomy and phylogeny of this much debated group.

The Use of mtDNA for the Identification of Plant Parasitic Nematodes

Mitochondria from all organisms studied to date have their own mitochondrial DNA (mtDNA), distinct from that of the nucleus. Animal mtDNA carries the genetic information for two rRNAs, a complete set of tRNAs and only a few essential components of the respiratory complexes: three subunits of the cytochrome oxidase (COI, COII and COIII), the cytochrome b (Cyt b), the subunits 6 and 8 of the Fo ATPase complex and several subunits of the NADH dehydrogenase (ND1-ND6 and ND4L). In addition, each molecule contains a control region in which signals of DNA duplication and transcription are included (for a review see Wolstenholme, 1993).

Molecular diagnostics and variability of longidorid nematodes

PCR-RFLP and sequencing approaches of ribosomal DNA are being used to study taxonomy, molecular identification and phylogeny of plant parasitic nematodes. In this paper, we discuss on the usefulness of ITS PCRRFLP analysis to differentiate among longidorid species. In addition, we examined how well ITS PCR-RFLP differentiated between longidorid species, and how well sequencing of two different ribosomal regions, the ITS containing region and D1-D2 domains of the 26S rDNA, were able to infer phylogenetic relationships among those same species. These methods and their advantages in identifying longidorids and establishing their phylogenetic relationships are examined and discussed.

Production and characterisation of monoclonal antibodies to antigens from Xiphinema index

When feeding, Xiphinema index induces remarkable cellular modifications at the feeding site in the root tips of its hosts, similar to those induced by root endoparasites such as Meloidogyne spp. The large size of X. index enables direct analysis of their proteins. Following immunisation of a mouse with macerated extracts of X. index bodies, a panel of monoclonal antibodies (MAbs) was obtained against this nematode. The MAbs were used in immunolocalisation studies and antibodies recognising epitopes present at the surface coat, inner cuticle, dorsal gland cell and dorsal duct, body wall muscle fibres, nervous system and reproductive system were identified. These antibodies provide useful tools for studying plant-nematode interactions.