D. J. F. Brown

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.

Transmission of tobacco rattle virus isolate PpK20 by its nematode vector requires one of the two non-structural genes in the viral RNA 2.

Tobacco rattle virus isolate PpK20 is transmitted by the nematode Paratrichodorus pachydermus. RNA 2 of the virus determines vector transmissibility and encodes the viral coat protein and two non-structural proteins with molecular masses of 29.4 kDa and 32.8 kDa. Deletions and a frameshift in the two non-structural genes did not interfere with encapsidation or co-replication of RNA 2 with RNA 1 after mechanical inoculation of plants. Mutations that affected the 29.4K gene or both non-structural genes abolished nematode transmission, whereas a large deletion in the 32.8K gene had no effect on transmission by P. pachydermus. It is concluded that the 29.4K gene but not the 32.8K gene is involved in transmission of isolate PpK20 by this vector.

Validation of the specificity and sensitivity of species-specific primers that provide a reliable molecular diagnostic for Xiphinema diversicaudatum, X. index and X. vuittenezi

Xiphinema diversicaudatum and X. index are vector nematode species of economic importance in viticulture regions as they can transmit Arabis Mosaic, Grapevine Fanleaf and Strawberry Latent Ringspot viruses to grapevine. Wang et al. (2003) designed species-specific diagnostic primers from ribosomal genes for both these vector species as well as a vector and a non-vector species X. italiae and X. vuittenezi, respectively. Our study aimed to confirm the specificity and determine the sensitivity and reliability of the primers for the two vector species, X. diversicaudatumand X. indexwhen challenged with closely related longidorid species and general nematode communities typical of vineyard soil. With one exception, no PCR product was observed when the primers were tested against six Longidorus, one Paralongidorus and one Xiphinema non-target species. Occasionally (three out of eight replicate PCR reactions) a weak PCR product was noted when primers for X. index were tested with L. elongatus. Furthermore, when challenged with a range of non-target nematode species comprising the nematode community typical of viticulture soil, no PCR product was amplified. An experimental dilution series of extracted DNA rigorously demonstrated that DNA from an equivalent single specimen of the target virus-vector species, X. diversicaudatum and/or X. index, could be detected amongst 1000 equivalent non-targetX. vuittenezi. Also, extracted DNA from an equivalent single target specimen was detected when added to DNA extracted from the overall soil nematode community. The primers were assessed further by using serial mixtures of actual nematodes rather than extracted DNA to simulate field soil. Using this method, a single target nematode could be detected amongst 200 non-target specimens. Given their specificity, sensitivity and reliability, it appears that these diagnostic primers will be of great benefit to phytosanitary/quarantine services related to the viticulture industry.

RNA-2 of tobacco rattle virus encodes the determinants of transmissibility by trichodorid vector nematodes.

Pseudorecombinant isolates produced from an efficiently transmitted and a non-transmitted isolate of tobacco rattle tobravirus were tested for their transmissibility by trichodorid vector nematodes. Isolates with RNA-2 originating from the non-transmissible isolate were not transmitted by the vector, whereas isolates with RNA-2 originating from the efficiently transmitted isolate were transmitted efficiently. It is therefore concluded that the factor determining vector transmissibility is located on the RNA-2 genome segment of TRV. Because the serological properties of the isolates also correlate with transmissibility, it is likely that the virus particle protein is involved in the transmission process.

Review of the ultrastructure of the nematode body cuticle and its phylogenetic interpretation

The phylogenetic interpretation of the nematode cuticle ultrastructure is reviewed within the framework of recent DNA‐sequence data. In particular, the structure of the median and basal zones is discussed. Several structural elements of the cuticle seem to have arisen independently several times within the Nematoda and thus are highly homoplasious (e.g. the cortical or basal radial striae, spiral fibre layers and a fluid matrix with struts). Moreover, identifying the homology of the nematode cuticle ultrastructures is often very difficult at deep taxonomic levels. Hence, the cuticle appears to be unreliable regarding resolution of deep‐level relationships in the Nematoda. However, at less inclusive taxonomic levels (e.g. families, genera, …) the cuticle seems to be a more reliable phylogenetic marker.

Sequence of RNA 2 of a nematode-transmissible isolate of tobacco rattle virus.

Tobacco rattle virus (TRV) isolate PPK20 is transmitted by Paratrichodorus pachydermus nematodes. The factor(s) determining vector transmissibility has been shown to be located on TRV RNA 2. Sequence determination revealed that PPK20 RNA 2 contains three open reading frames encoding the coat protein (cp) and proteins with molecular masses of 29.4 and 32.8 kDa. The 29.4 and 32.8 kDa protein-coding genes showed no significant sequence similarity to any other known tobravirus gene. A full-length cDNA of PPK20 RNA 2 cloned between the 35S promoter and nos terminator infected plants when co-inoculated with PPK20 RNA 1. Deletions in the reading frames of the 29.4 and 32.8 kDa proteins revealed that these sequences are dispensable for replication of PPK20 RNA 2 in plants. Subgenomic RNAs for translation of cp and the putative 29.4 and 32.8 kDa proteins were detected in infected leaves. The possible role of PPK20 RNA 2 non-structural genes in TRV vector transmission is discussed.

Development and validation of species-specific primers that provide a molecular diagnostic for virus-vector longidorid nematodes and related species in German viticulture

The nematode species Longidorus attenuatus, L. elongatus, L. macrosoma and Paralongidorus maximusare economically important pests to the viticulture industry due to their ability to vector two nepoviruses (Raspberry Ringspot Virus and Tomato Black Ring Virus) to grapevines. In Germany, these species occur in vineyard soil with other non-vector but morphologically similar longidorid species, L. helveticus, L.␣profundorum and L. sturhani. Species-specific primers were designed from ribosomal DNA for all seven species to facilitate taxonomic identification for non-specialists. Primers were assessed for their reliability by screening, where possible, a number of populations of each species. Furthermore, their selectivity and sensitivity were determined when challenged with closely related longidorid species and general nematode communities typical of vineyard soil. A multiplex approach using a common forward primer combined with species-specific reverse primers enabled three target nematode species to be detected in the same PCR reaction. All primers were highly specific, detecting all nematode developmental forms from disparate populations and were sufficiently sensitive to detect a single target nematode within a whole nematode community typical of a vineyard soil comprising of a range of non-target species. Given their specificity, sensitivity and reliability, these diagnostic primers should be of great benefit to both phytosanitary/quarantine services related to the viticulture industry and also as a decision management tool for growers.

Analysis of expressed sequence tags from the ectoparasitic nematode Xiphinema index

We report the results of a small-scale expressed sequence tag project performed on the ectoparasitic nematode Xiphinema index. Approximately 1400 genes were sequenced, 70% from a cDNA library generated from dissected basal bulbs (containing the pharyngeal gland cells) and 30% from cDNA libraries generated from whole mixed stage nematodes. A large portion of the bulb library (48%) was composed of proteins with no matches in the database. Further analysis of these genes revealed that a total of 51 contigs were present, half of which encoded novel secreted proteins. By contrast, the whole nematode library contained more housekeeping and nematode-specific genes. Only one of the novel genes from the whole nematode library had a predicted signal peptide at the N-terminus. Genes encoding transthyretin-like proteins were abundant in the bulb library and in situ hybridisation confirmed that one of these is expressed in the basal bulb. Genes encoding a variety of proteases, which were shown using in situ hybridisation to be expressed in the gut, were also identified.

Longidorus Arthensis Sp. N. (Nematoda: Longidoridae) a Vector of Cherry Rosette Disease Caused By a New Nepovirus in Cherry Trees in Switzerland

A new Longidorus species was found associated with cherry rosette disease (CRD), caused by an uncharacterised nepovirus, in cherry trees in Switzerland. Longidorus arthensis sp. n. is characterised by having a wide bluntly rounded, lip region almost continuous with the body contour, large evenly bilobed amphidial pouches, bluntly rounded tail, common, functional males and first stage juveniles with a pegged tail terminus. Populations of L. arthensis were recovered from four cherry orchards, in the Arth region of Switzerland, in which some trees showed CRD symptoms. Bait testing individual nematodes collected from all four orchards confirmed L. arthensis as the natural vector of a nepovirus causing CRD. Largest numbers of L. arthensis were recovered from the deeper soil layers, below 30 cm, therefore to optimise detection of the nematode, soil samples should be collected below 30 cm depth.

Comments On the Taxonomy, Occurrence and Distribution of Longidoridae (Nematoda) in North America

The occurrence, distribution and associations with nepoviruses of members of the Longidoridae in North America are presented for each species. Three genera of Longidoridae, Longidorus (10 species), Paralongidorus (2 species), and Xiphinema (38 species) have been reported from North America. Twenty of the Xiphinema species are from the X. americanum group, twelve species have a conoid digitate tail, six species have a hemispherical tail with or without a peg, and two have a long conical tail. Seven North American Xiphinema species have a Z-differentiation and five have only one ovary. One species, X. krugi, has a normal posterior genital branch and ovary and a reduced anterior genital branch lacking an ovary. Longidorus diadecturus differs from all other North American Longidorus species in having weakly developed odontophore flanges and a guide-ring at about mid-odontostyle. Many of the species identifications require to be confirmed, especially those within the X. americanum-group. Five Xiphinema and one Longidorus species are natural vectors of nepoviruses in North America. It is suggested that methods routinely used in North American laboratories for extracting nematodes from soil are inappropriate for recovering Longidoridae and that these nematodes are more widespread and occur more frequently than indicated in published literature.