Gerrit Karssen

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 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.

Mitochondrial coding genome analysis of tropical root-knot nematodes (Meloidogyne) supports haplotype based diagnostics and reveals evidence of recent reticulate evolution.

The polyphagous parthenogenetic root-knot nematodes of the genus Meloidogyne are considered to be the most significant nematode pest in sub-tropical and tropical agriculture. Despite the crucial need for correct diagnosis, identification of these pathogens remains problematic. The traditionally used diagnostic strategies, including morphometrics, host-range tests, biochemical and molecular techniques, now appear to be unreliable due to the recently-suggested hybrid origin of root-knot nematodes. In order to determine a suitable barcode region for these pathogens nine quickly-evolving mitochondrial coding genes were screened. Resulting haplotype networks revealed closely related lineages indicating a recent speciation, an anthropogenic-aided distribution through agricultural practices, and evidence for reticulate evolution within M. arenaria. Nonetheless, nucleotide polymorphisms harbor enough variation to distinguish these closely-related lineages. Furthermore, completeness of lineage sorting was verified by screening 80 populations from widespread geographical origins and variable hosts. Importantly, our results indicate that mitochondrial haplotypes are strongly linked and consistent with traditional esterase isozyme patterns, suggesting that different parthenogenetic lineages can be reliably identified using mitochondrial haplotypes. The study indicates that the barcode region Nad5 can reliably identify the major lineages of tropical root-knot nematodes.

Description of Pratylenchus dunensis sp. n. (Nematoda: Pratylenchidae), a root-lesion nematode associated with the dune grass Ammophila arenaria (L.) Link

Summary – A root-lesion nematode, Pratylenchus dunensis sp. n., is described and illustrated from Ammophila arenaria (L.) Link, a grass occurring abundantly in coastal dunes of Atlantic Europe. The new species is characterised by medium sized (454-579 µm) slender, vermiform, females and males having two lip annuli (sometimes three to four; incomplete incisures only visible with scanning electron microscopy), medium to robust stylet (ca 16 µm) with robust stylet knobs slightly set off, long pharyngeal glands (ca 42 µm), lateral field with four parallel, non-equidistant, lines, the middle ridge being narrower than the outer ones, lateral field with partial areolation and lines converging posterior to the phasmid which is located between the two inner lines of the lateral field in the posterior half of the tail, round spermatheca filled with round sperm, vulva at 78% of total body length and with protruding vulval lips, posterior uterine sac relatively short (ca 19 µm), cylindrical tail (ca 33 µm) narrowing in the posterior third with smooth tail tip and with conspicuous hyaline part (ca 2 µm). Males occur abundantly and present similar characteristics except for smaller dimensions for all morphological characters, but the head region is more truncated in outline than the female, spicule length is ca 15 µm and testis length is ca 195 µm. Nucleotide sequences of the rDNA expansion region D2D3 differed from the morphologically similar species P. penetrans and P. brzeskii that also occur in coastal dunes. These differences are supported by PCR-RFLP of the ITS-rDNA. Pratylenchus dunensis sp. n. was also found parasitising roots of Elymus farctus Viv.

On the species status of the root-knot nematode Meloidogyne mayaguensis Rammah & Hirschmann, 1988

Abstract Holo- and paratypes of the root-knot nematodes Meloidogyne mayaguensis Rammah & Hirschmann, 1988 and Meloidogyne enterolobii Yang & Eisenback, 1983 were morphometrically and morphologically compared. All observed female, male and second-stage juvenile morphometrical and morphological characters are identical for the two studied species. Additionally, contradictions between the original species descriptions were unravelled. The present study of holo- and paratypes confirms the taxonomical status of Meloidogyne mayaguensis as a junior synonym for Meloidogyne enterolobii.

First Report of the Root-Knot Nematode Meloidogyne ethiopica on Tomato in Slovenia

The root-knot nematode Meloidogyne ethiopica Whitehead originally described from Tanzania is also distributed in South Africa, Zimbabwe, and Ethiopia (3). Although this species is a relatively unknown root-knot nematode, M. ethiopica parasitizes several economical important crops, such as tomato, cowpea, bean, cabbage, pepper, pumpkin, tobacco, lettuce, and soybean (4). This species can be morphologically confused with M. incognita (Kofoid & White), and therefore, is probably easily overlooked (1). Recently, characteristic esterase isozyme patterns were described for this species, which provide a more reliable identification (2). In October 2003, Lycopersicum esculentum cv. Belle plants with large root-galls were observed in a greenhouse at Dornberk, Slovenia, which were identified as an unknown root-knot nematode within the M. incognita group. Subsequent sampling revealed infestation of all tomato plants within this greenhouse with obvious aboveground symptoms of stunting and wilting. Additional morphological and isozyme studies identified this root-knot nematode as M. ethiopica. The identification was based on female, male, and second-stage juvenile morphology and malate dehydrogenase and esterase isozyme phenotypes from young egg-laying females isolated from 20 tomato plants and compared with a culture of M. ethiopica from Africa. To our knowledge, this is not only the first report of M. ethiopica in Slovenia, but also the first report of this species in Europe. References: (1) A. M. Golden. Fundam. Appl. Nematol. 15:189, 1992. (2) W. Mandefro and K. Dagne. Afr. J. Plant Prot. 10:39, 2000. (3) A. G. Whitehead. Trans. Zool. Soc. Lond. 31:263, 1968. (4) A. G. Whitehead. Nematologica 15:315, 1969.

Phylogeny and Evolution of Nematodes

Many plant-parasitic nematodes including members of the genera Meloidogyne (root-knot nematodes), Heterodera and Globodera (cyst nematodes) and Pratylenchus (lesion nematodes) are studied as they cause major damage to crops such as potato, tomato, soybean and sugar beet. Both for fundamental reasons (Can we reconstruct the evolution of these parasites in the almost complete absence of fossil records?) and for practical reasons (Can we use phylogenetic data to detect specific parasitic species in pools of mainly harmless/beneficial nematodes?) it is important to get an insight into the relationship between these plant-parasitic nematodes and their fungivorous and bacterivorous relatives. In this chapter we will summarize and comment on the rapid increase of molecular (mainly ribosomal DNA) data published over the last decade. The greatest attention will be paid to the Tylenchomorpha (Clade 12), and the relationship within and between root lesion and root-knot nematodes (the genera Pratylenchus and Meloidogyne).

Radopholus arabocoffeae sp. n. (Nematoda: Pratylenchidae), a nematode pathogenic to Coffea arabica in Vietnam, and additional data on R. duriophilus

Radopholus arabocoffeae sp. n., a new nematode pathogenic on Coffea arabica cv. Catimor, is described from Vietnam. Females of R. arabocoffeae sp. n. are characterised by the broad amphidial apertures with prominent margins. Males are characterised by the bursa extending to one third, rarely middle, of the tail. The new species belongs to the group of species with a long tail in the female. Radopholus arabocoffeae sp. n. is easily distinguished from R. similis by the bursa reaching to only one third of the tail vs extending to the tail terminus. Radopholus arabocoffeae sp. n. is differentiated from R. bridgei by the lateral field having three bands of equal width (vs middle one narrower than others), lateral field completely areolated over whole body vs not areolated except irregularly in neck and tail, hemizonid distinct vs indistinct, four lateral field incisures terminating far behind phasmid vs three incisures terminating at or just behind phasmid, lateral lines fusing at two thirds of tail vs fusing at one third of tail, longer spicule length (18-21 vs 15.5-18.0 mum), and male bursa usually extending to only one third of tail vs mid tail. Radopholus arabocoffeae sp. n. differs from R. colbrani by the rod-like vs round sperm, spicule length (18-21 vs 13-16 mum), tail length to stylet ratio (4.1-4.9 vs more than 5.1) and presence vs absence of a bursa. Radopholus arabocoffeae sp. n. differs from R. duriophilus by the rod-like vs kidney-shaped sperm. Males further differ from R. duriophilus by shorter stylet length (8.2-11.6 vs 11.5-15 mum), smaller distance between dorsal pharyngeal gland orifice and stylet base (1.7-3.4 vs 4-9.5 mum), shorter hyaline tail (2.6-3.4 vs 4-9.5 mum), and bursa extending to one third of tail vs mid-tail. Female R. arabocoffeae sp. n. differ from R. duriophilus by the broad amphidial aperture with prominent margin present vs absent. Males of R. arabocoffeae sp. n. differ from R. musicola by the rudimentary and amalgamated stylet base (vs with knobs), and inner lateral lines fusing at two thirds of the tail vs just posterior to the phasmid. The high level of ITS-rDNA sequence divergence of R. arabocoffeae sp. n. from other Radopholus spp. and the presence of nucleotide autapomorphies support a separate specific status of this new species. On carrot disks, the two species reproduced from 15-30°C; optimum reproduction occurring at 28°C. The reproductive capacity of R. duriophilus was higher than that of R. arabocoffeae sp. n. Radopholus duriophilus reproduced from single juveniles; R. arabocoffeae sp. n. did not. The correlation between initial densities of Pratylenchus coffeae, R. duriophilus and R. arabocoffeae sp. n. and the weight of C. arabica cv. Catimor fitted the Seinhorst model Y = ym for Pi ge T, and Y = ym · m + ym(1–m)z(Pi–T). Coffea arabica cv. Catimor was very susceptible for to all three nematode species tested, but especially so to R. arabocoffeae sp. n. The reproductive capacity of R. arabocoffeae sp. n. on C. arabica cv. Catimor was higher than P. coffeae or R. duriophilus.

Root-knot nematode perineal pattern development: a reconsideration

The perineal pattern, a unique and complex structure located at the female posterior body region, comprises the vulva-anus area, tail terminus, phasmids, lateral lines and surrounding cuticular striae. Little is known about the development of this important but variable character complex. Published information together with scanning electron and light microscopical observations on female development and perineal patterns were used to explain in detail the perineal pattern. The basic pattern, with fine striae, is formed just after the last moult when the vulva is induced above the anus at the posterior ventral body side. This process is likely to be influenced by the previous feeding stage. The vulva-anus region moves posteriorly, while the female is still enclosed by the old cuticle layers. After resuming feeding, the female diameter increases rapidly and pattern striae become folded and coarser. The role of the expanding rectal glands and perineal pattern structures as lateral lines, anus, tail terminus and punctations are discussed in detail. Major origins for pattern variation are developmental and aging factors in both feeding stages, a vulva-anus shift and the observation of patterns of different age, in combination with variable interpretation and description.

The cellular structure of the female reproductive system within the Heteroderinae and Meloidogyninae (Nematoda)

Gonads from living young females, representing 23 different species, were extracted to study the cellular structure of the female genital structure within the Meloidogyninae and Heteroderinae. All genera studied can be characterised by their cellular spermatheca morphology. Within Meloidogyne a spherical spermatheca is found with lobe-like protruding cells, most species having 16 to 18 spermatheca cells with interlaced cell boundaries while M. microtyla and M. ichinohei have more spermatheca cells with different cell boundaries. Heterodera and Globodera reveal a comparable gonad structure. The spermatheca cells of Heterodera are columnar and arranged in a restricted number of rows, whereas in Globodera the spermatheca cells are squarish to rounded, depending on the species. The gonad morphology of Afenestrata koreana is clearly different from what would be expected based on the related genera Globodera and Heterodera. The apparently simplest genital system was found in Meloidodera floridensis where the uterus has a limited number of cells. In the other genera studied a large and variable cell number was found.