S.J.J. van den Elsen

A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences

As a result of the scarcity of informative morphological and anatomical characters, nematode systematics have always been volatile. Differences in the appreciation of these characters have resulted in numerous classifications and this greatly confuses scientific communication. An advantage of the use of molecular data is that it allows for an enormous expansion of the number of characters. Here we present a phylogenetic tree based on 1215 small subunit ribosomal DNA sequences ( ca 1700 bp each) covering a wide range of nematode taxa. Of the 19 nematode orders mentioned by De Ley et al. (2006) 15 are represented here. Compared with Holterman et al. (2006) the number of taxa analysed has been tripled. This did not result in major changes in the clade subdivision of the phylum, although a decrease in the number of well supported nodes was observed. Especially at the family level and below we observed a considerable congruence between morphology and ribosomal DNA-based nematode systematics and, in case of discrepancies, morphological or anatomical support could be found for the alternative grouping in most instances. The extensiveness of convergent evolution is one of the most striking phenomena observed in the phylogenetic tree presented here – it is hard to find a morphological, ecological or biological characteristic that has not arisen at least twice during nematode evolution. Convergent evolution appears to be an important additional explanation for the seemingly persistent volatility of nematode systematics.

Small Subunit rDNA-Based Phylogeny of the Tylenchida Sheds Light on Relationships Among Some High-Impact Plant-Parasitic Nematodes and the Evolution of Plant Feeding

Cyst (Heteroderidae), root knot (Meloidogyne spp.), and lesion (Pratylenchus spp.) nematodes all belong to a single nematode order, Tylenchida. However, the relationships between and within these economically highly relevant groups, and their relatedness to other parasitic Tylenchida is unclear. We constructed a phylogeny of 116 Tylenchida taxa based on full length small subunit ribosomal DNA (small subunit [SSU] rDNA) sequences. Ancestral state reconstruction points at a gradual development of simple to more complex forms of plant parasitism. Good resolution was observed in distal clades that include cyst, root knot, and lesion nematodes, and monophyly of most families was confirmed. Our data suggest that root knot nematodes have evolved from an ancestral member of the genus Pratylenchus, but it remains unclear which species is closest to this branching point. Contrary to the notoriously polyphagous distal representatives, basal members of the genus Meloidogyne (and probably, their common ancestor) have narrow host ranges. Our analysis also shows that mitotic parthenogeny has arisen at least two times independently among root knot nematodes. In many cases resolution till species was observed, suggesting that SSU rDNA sequences have a potential for DNA barcode-based species identification with, due to the overall conserved nature of this gene, limited intra-species variation.

Small Subunit Ribosomal DNA-Based Phylogenetic Analysis of Foliar Nematodes (Aphelenchoides spp.) and Their Quantitative Detection in Complex DNA Backgrounds

Foliar nematodes, plant-parasitic representatives of the genus Aphelenchoides, constitute a minority in a group dominated by fungivorous species. Distinction between (mostly harmless) fungal feeding Aphelenchoides species and high impact plant parasites such as A. besseyi, A. fragariae, A. ritzemabosi, and A. subtenuis is severely hampered by the scarcity of informative morphological characters, some of which are only observable in specific developmental stages. Poor description of a number of non-plant-parasitic Aphelenchoides species further complicates identification. Based on (nearly) full-length small subunit ribosomal DNA (SSU rDNA) sequences (≈1,700 bp), a phylogenetic tree was generated, and the four target species appeared as distinct, well-supported groups. Notably, this genus does not constitute a monophyletic group: A. besseyi and A. ritzemabosi cluster together and they are phylogenetically isolated from A. fragariae, A. subtenuis, and most other fungivorous species. A phylum-wide SSU rDNA framework was used to identify species-specific DNA motifs. For the molecular detection of four plant-parasitic Aphelenchoides species, polymerase chain reaction primers were developed with high, identical annealing temperatures (63°C). Within the molecular framework presented here, these primers can be used for the rapid screening of plant material and soil for the presence of one or multiple foliar nematode species.

Both SSU rDNA and RNA polymerase II data recognise that root-knot nematodes arose from migratory Pratylenchidae, but probably not from one of the economically high-impact lesion nematodes

In 2000 Siddiqi formulated a hypothesis stating that root-knot nematodes (Meloidogyne spp.) constitute a branch arising from yet another important group of plant parasites, the migratory Pratylenchidae. This hypothesis was solely based on morphological characteristics. Ribosomal DNA (rDNA) sequence analysis supports this hypothesis in its broad sense, but the more precise question about the identity of a migratory Pratylenchidae representative being closest to the most basal Meloidogyne species could not be addressed due to a lack of backbone resolution (Holterman et al., 2009). Here we present an extended small subunit rDNA sequence analysis and a data set of partial RNA polymerase II sequences from Pratylenchidae and basal Meloidogynidae. Our data point at members of the genus Pratylenchus as being closest to the common ancestor of the root-knot nematodes, but it was not possible unequivocally to identify a candidate lesion nematode species. Pratylenchus is a species-rich genus (ca 70 valid species), and we suggest that the species closest to the most basal root-knot nematode should be sought outside of the group of relatively well-characterised, agronomically relevant, species.

A rapid, sensitive and cost-efficient assay to estimate viability of potato cyst nematodes

Potato cyst nematodes (PCNs) are quarantine organisms, and they belong to the economically most relevant pathogens of potato worldwide. Methodologies to assess the viability of their cysts, which can contain 200 to 500 eggs protected by the hardened cuticle of a dead female, are either time and labor intensive or lack robustness. We present a robust and cost-efficient viability assay based on loss of membrane integrity upon death. This assay uses trehalose, a disaccharide present at a high concentration in the perivitelline fluid of PCN eggs, as a viability marker. Although this assay can detect a single viable egg, the limit of detection for regular field samples was higher, ≈10 viable eggs, due to background signals produced by other soil components. On the basis of 30 nonviable PCN samples from The Netherlands, a threshold level was defined (ΔA(trehalose) = 0.0094) below which the presence of >10 viable eggs is highly unlikely (true for ≈99.7% of the observations). This assay can easily be combined with a subsequent DNA-based species determination. The presence of trehalose is a general phenomenon among cyst nematodes; therefore, this method can probably be used for (for example) soybean, sugar beet, and cereal cyst nematodes as well.

First Report of Plant-Parasitic Nematode Meloidoderita salina in the Netherlands

After the description of the root-parasitic nematode Meloidoderita salina from a tidal salt marsh in France (1), an additional sampling was carried out to search for the presence of this unusual nematode in a tidal salt marsh area close to Sint-Annaland, Zeeland Province, the Netherlands. In August and October 2012, a total of 25 soil and root samples were collected from the halophytic plants Atriplex portulacoides L. (so far the only known host for this nematode species), A. littoralis L., A. prostrata Boucher ex DC., Limonium vulgare Mill., Salicornia europaea L., Aster tripolium L., and Plantago maritima L. All these halophytes grow in a cohesive muddy soil type within the salt marsh, except A. littoralis and A. prostrata, which grow in the litter tidal zones on the edges of this area. Nematodes from roots and soil were extracted by centrifugal flotation (2) and Oostenbrinks cotton-wool filter methods (4), respectively. Additionally, roots were used for direct observation of females and young cystoids with a dissecting microscope. Finally, all stages were compared morphologically with available type material (1). Root and soil samples demonstrated that only nematodes isolated from A. portulacoides, A. littoralis, and A. prostrata contained all life stages of the genus Meloidoderita, while on the roots of L. vulgare, S. europaea, A. tripolium, and P. maritima, no Meloidoderita was observed. The soil samples included males, cystoids, and second-stage juveniles (J2) in low densities (<20 nematodes/100 ml), while swollen females and young cystoids were observed on root samples. All stages (n = 10 per life stage) fit morphologically with the recently described M. salina. Females were swollen with an oval to pear shaped body with a small posterior protuberance, irregular and twisted neck, oval and backwardly sloping stylet knobs, a prominent secretory-excretory (S-E) pore with cuticular lobes, and a swollen uterus with a thick hyaline wall. Males were without stylet, strongly sclerotized S-E duct, and tail tapering to rounded terminus ending in one or two ventrally terminal mucron. J2s had a well-developed stylet and rounded knobs set off from shaft and conical tail slightly curved ventrally and tapering to a finely pointed terminus with a finger-like projection. Cystoids showing the unique sub-cuticular hexagonal beaded pattern (1). J2s were also used for molecular analysis. DNA was extracted by incubating individual J2s in a lysis buffer as described in (3). Two primer combinations were used to amplify the small subunit ribosomal DNA (SSU rDNA) from a 100-times-diluted crude lysate (two overlapping fragments, [3]). The resulting (nearly) full-length SSU-rDNA sequences (GenBank KF751617 and KF751618) showed >99% identity with M. salina sequences from nematodes collected in the aforementioned tidal salt marsh in France (FJ969126 and FJ969127). To our knowledge, this is the first report of M. salina in the Netherlands. Moreover, this is the first record of M. salina parasitizing A. littoralis and A. prostrata. Although these Atriplex species are used for human consumption, the effect of M. salina on the host is unknown so far. References: (1) S. Ashrafi et al. Zookeys 249:1, 2012. (2) W. A. Coolen. Pages 317-329 in: Root-knot nematodes (Meloidogyne species). Systematics, biology and control. Academic Press, New York, 1979. (3) Holterman et al. Mol. Biol. Evol. 23:1792, 2006. (4) M. Oostenbrink. Pages 85-102 in: Nematology. University of North Carolina Press, Chapel Hill, 1960.

Morphological and molecular data support the monophyletic nature of the genus Pratylenchoides Winslow, 1958 (Nematoda: Merliniidae) and reveal its intrageneric structuring

The genus Pratylenchoides has recently been transferred from the family Pratylenchidae to Merliniidae. To investigate further the relationship between these ‘Pratylenchus-like’ species (residing in the subfamily Pratylenchoidinae) and the subfamily Merliniinae, more than 500 soil samples were collected from various natural and agronomic habitats in the northern and north-western provinces of Iran. In this study, paratypes or populations of 22 species of Pratylenchoides, including the Iranian populations of P. alkani, P. crenicauda, P. erzurumensis, P. laticauda, P. nevadensis, P. ritteri and an undescribed species, were studied. Intra- and interspecies variation of the following characters were investigated: position of the pharyngeal gland nuclei, shape of female and male head, striation of female tail terminus, number of lateral lines at mid-body and in phasmid region for females, presence of intestinal fasciculi, and shape of sperm. Combining morphological and molecular data prompted us to propose two clusters of related Pratylenchoides species. One cluster includes P. crenicauda, P. variabilis and P. erzurumensis, whereas the second cluster consists of P. alkani, P. nevadensis and P. ritteri. Our data point to a sister positioning of P. magnicauda vis-a-vis all Pratylenchoides species included in this research. Analyses of SSU rDNA (for family and subfamily relationships) and partial LSU rDNA sequences (for intrageneric relationships) data revealed: i) the distal and nested positioning of all Pratylenchoidinae within the Merliniidae; ii) the single transition from ectoparasitism to migratory endoparasitism within the family Merliniidae corresponds with the current subfamily partitioning; and iii) support for the monophyletic nature of the genus Pratylenchoides.