Eduardo DE LA PEÑA 1,, Maurice MOENS 1,2, Adriaan VAN AELST 3 and Gerrit KARSSEN 4,5

Transcription

1 Nematology, 2006, Vol. 8(1), Description of Pratylenchus dunensis sp.n.(nematoda: Pratylenchidae), a root-lesion nematode associated with the dune grass Ammophila arenaria (L.) Link Eduardo DE LA PEÑA 1,, Maurice MOENS 1,2, Adriaan VAN AELST 3 and Gerrit KARSSEN 4,5 1 Agricultural Research Centre, Crop Protection Department, Burg. van Gansberghelaan 96, 9820, Merelbeke, Belgium 2 Gent University, Laboratory for Agrozoology, Coupure 653, 9000 Gent, Belgium 3 Wageningen University & Research Centre, Laboratory of Plant Cell Biology, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands 4 Plant Protection Service, Nematology Section, P.O. Box 9102, 6700 HC Wageningen, The Netherlands 5 Wageningen University & Research Centre, Laboratory of Nematology, Binnenhaven 5, 6709 PD Wageningen, The Netherlands Received: 4 April 2005; revised: 7 November 2005 Accepted for publication: 7 November 2005 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 ( 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. Keywords D2D3, description, Elymus farctus, ITS-rDNA, molecular, morphology, morphometrics RFLP. Plant-parasitic nematodes and their influence on plant competition in semi-natural and natural systems such as coastal dunes have received considerable attention in the past few years (De Rooij-Van der Goes et al., 1995; Van der Putten & Peters, 1997; Verschoor et al., 2002). Two species of the genus Pratylenchus Filipjev, 1936, viz P. brzeskii Karssen, Waeyenberge & Moens, 2000 and P. penetrans (Cobb, 1917) Filipjev & Schuurmans Stekhoven, 1941, have been reported on Ammophila arenaria (L.) Link and A. breviligulata Fern., respectively, and are thought to be involved in the die-out of these plants in coastal dunes (Seliskar & Huettel, 1993; Karssen et al., 2000). In 1998, specimens of the genus Pratylenchus were detected in the roots and rhizosphere of A. arenaria sampled in the dune area of Groote Keeten, province Noord- Holland, The Netherlands. Although the population differed from P. penetrans by the number of lip annules, tail morphology and some morphometrics, it was identified as P. penetrans because the ITS-RFLP of the population were identical to those of P. penetrans as published by Waeyenberge et al. (2000). As a result, P. penetrans was Corresponding author, e.delapena@clo.fgov.be Koninklijke Brill NV, Leiden, Also available online -

2 E. de la Peña et al. reported in coastal dunes of The Netherlands for the first time (Karssen et al., 2001). During 2002 and 2003, nematode surveys were made in dune areas with vigorous A. arenaria stands along the European Atlantic coast. Once again, specimens similar to P. penetrans but showing the above mentioned deviations in morphology and morphometrics were detected at different sampling sites. Their D2D3 LSU sequences showed further differences from those of P. penetrans populations. As a consequence the identity of the population from Groote Keeten was reconsidered and DNA sequence comparisons with more P. penetrans populations indicated that the differences were too striking to be taken as intraspecific variation. Therefore, more comparisons of D2D3 sequences and eventually also of ITS-RFLP, as made previously, were combined with extra observations on the morphology and morphometrics. The additional data revealed that the population belongs to a new species, which in this paper is described as Pratylenchus dunensis sp. n. Materials and methods NEMATODE POPULATIONS Pratylenchus dunensis sp. n. was isolated from soil and roots collected in the rhizosphere of A. arenaria in the coastal dunes of Groote Keeten (Province Noord-Holland, The Netherlands). The new species was compared with a population of P. penetrans isolated from Wisteria sinensis Sweet in Maaseik (Belgium) and a population of P. brzeskii collected on A. arenaria in Biarritz (France). Females and males were extracted from the roots in a mist chamber (Seinhorst, 1950). MORPHOLOGICAL STUDY For light microscopical observations, males and females were fixed in TAF (Courtney et al., 1955). Nematodes were mounted in anhydrous glycerine using the slow method of Hooper and Evans (1993). Morphometrics were taken using an Olympus BX50 compound microscope equipped with Leica image-capture IM500 system and software. For scanning electron microscopy (SEM), fixed nematodes were coated with 45 nm of platinum in a preparation chamber (CT-1500HT, Oxford Instruments, High Wycombe, UK) and observed with a Jeol 6300F field emission electron microscope at 3.5 kv. MOLECULAR STUDIES For each of the nematode species, DNA was extracted from previously identified female nematodes. Each nematode was placed in 25 µl digestion buffer (100 mm KCl, 20 mm Tris ph 8, 3 mm MgCl 2, 2 mm DTT, 0.9% Tween 20) diluted with 25 µl water and proteinase K (600 µg/ml). The extract was incubated at 65 Cfor2h and then 5 min at 95 C to denaturise proteinase K. The crude extract was kept at 70 C until use. Five µl ofthe crude extract was used for PCR amplification. Primers and PCR conditions for both the D2D3 and the ITS (including the 5.8S gene and flanking areas of the 18S and 28S genes of rdna) amplification were as described by De Ley et al. (1999) and Waeyenberge et al. (2000), respectively. After electrophoresis in 1% TAE-buffered agarose gels (1 h, 100 V), the PCR product was visualised under UV light. DNA was excised from the gels using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany), cloned into pgem-t vector and transformed into JM109 High Efficiency Competent Cells (Promega, Leiden, The Netherlands). Several clones were isolated using blue/white selection and sequenced using vector primers with a BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA, USA). The resulting products were purified using a Centriflex Gel Filtration Cartridge (Edge Biosystems, Gaithersburg, MD, USA). DNA samples were sequenced by ABI Prism 377 DNA Sequencer (PE Applied Biosystems). DNA sequences were edited manually with Chromas 1.45 (Technelysium, Helensvale, Australia), and aligned with Clustal X 1.64 (default options) (Thompson et al., 1997). The sequences of Pratylenchus dunensis sp.n.aredeposited in EMBL ( under the accession numbers AJ890459, AJ890460, AJ and AJ890462; the P. brzeskii sequence was deposited under the accession number AJ Differences between sequences were estimated using the DNA distance option provided by BioEdit sequence alignment editor (Hall, 1999). Amplified PCR products were purified using a Qiagen Gel Purification Kit. After purification, one µl of purified PCR product was digested with one of five restriction enzymes, viz., CfoI, HindIII, PstI, EcoRI and MspI, according to the instructions of the manufacturer (Amersham Bioscience, Little Chalfont, UK). 80 Nematology

3 A new species of Pratylenchus from dune grass Pratylenchus dunensis * sp. n. = P. penetrans apud Karssen et al., 2001 (Figs 1-4) MEASUREMENTS See Table 1. DESCRIPTION Female Body vermiform, slender and tapering towards both ends. Cuticle finely annulated with annules µm wide at mid-body. Head slightly set-off, three times height in diam., rounded with prominent cephalic sclerotisation and vestibule extension. Two lip annuli, often with one or two incomplete, transverse incisures not visible with light microscope. Labial disc oval, elevated, fused with four submedial lips forming smooth, dumb-bell shaped, headcap. Cephalic sensilla marked by subtle depression on each submedial lip. SEM en face view showing oval prestoma. Stylet finely shaped, relatively short, with robust, anteriorly indented, set-off knobs. Stylet cone length equal or shorter than length of shaft plus knobs. Hemizonid near isthmus level, 2-3 µm in length, anterior to oval shaped secretory-excretory pore located between nerve ring and pharyngeal junction. Pharyngeal gland lobe three body diam long. Vulva with well-developed lips, often protruding. Lateral field with four lateral lines starting posterior to stylet; occupying ca one-third of body diam. at mid-body. At pharyngeal-vulva region, middle ridge narrower than outer ones; at mid-body, width of inner ridge 60-80% of distance between outer lines. Outer incisures partially areolated, with striae crossing outer lateral field ridges every µm. Lateral lines converging posterior to phasmid. Genital tract monoprodelphic with single row of oocytes. Spermatheca round to slightly oval, filled with rounded sperm. Post-uterine sac undifferentiated, short. Tail cylindrical with distinct annulations, narrowing in posterior third with smooth, dome-shaped, terminus; some specimens (one in five of adult females) with one or two indentations next to smooth tail tip. Hyaline part distinct. Anus round to oval shaped. Small, rounded, phasmid located between inner lateral field lines, posterior to mid-tail. * Specific epithet derived from the dune habitat. Male Occurring abundantly (nearly 50% of adults). Morphologically similar to females, but smaller for all non-sexual characters. Head characters as in females, but more truncated in outline. Apical annules slightly raised. Incomplete transverse incisures and variation in lip annuli as described for females. Single testis anteriorly outstretched. Spicules and gubernaculum ventrally curved. Bursa enclosing tail. Ventral side of bursa coarsely crenate; phasmidial orifice on bursa located at almost mid distance between cloaca and tail tip. TYPE HOST AND LOCALITY Pratylenchus dunensis sp. n. was found in roots of Ammophila arenaria growing in the fore dunes of Groote Keeten, Province Noord Holland, The Netherlands. The species was also detected in the roots of Elymus farctus Viv. present at the same area. The distribution of P. dunensis sp. n. seems to be favoured by conditions in front dunes where there is considerable sand deposition and the two dune grasses grow vigorously. In older, more stabilised, dunes this species is less abundant. OTHER LOCALITIES AND HOSTS Pratylenchus dunensis sp. n. has also been found in Oostvoorne, The Netherlands, parasitising Ammophila arenaria and Elymus farctus roots, and in fore dunes at Blakeney Point, Norfolk, UK on A. arenaria. TYPE MATERIAL One holotype female, six female and seven male paratypes at the Nematode Collection of Wageningen (collection numbers WT3399 and WT3400), University and Research Centre, Wageningen, The Netherlands. Eight male and eight female paratypes at the Museum voor Dierkunde (collection numbers UGMD and UGMD ), University of Gent, K.L. Ledeganckstraat 35, 9000 Gent, Belgium. DIAGNOSIS AND RELATIONSHIPS Pratylenchus dunensis sp. n. is characterised by two lip annuli with incomplete transverse incisures, a stylet (ca 16 µm) with robust, anteriorly indented and set off knobs, vulva at 78% of body length, lateral field with four parallel lines unequally spaced from pharynx to vulva, the inner ridge occupying 60-80% of the distance Vol. 8(1),

4 E. de la Peña et al. Fig. 1. Pratylenchus dunensis sp. n. A: Entire female body, lateral view; B: Entire male body, lateral view; C: Male cephalic region; D: Female cephalic region; E: Female anterior end including pharyngeal gland; F: Female vulval region, ovary and spermatheca; G: Lateral field at mid-body; H: Female tail tips; I: Female posterior end; J: Male posterior end. 82 Nematology

5 A new species of Pratylenchus from dune grass Table 1. Morphometric characters of Pratylenchus dunensis n. sp. Measurements are in µm and in the form: mean ± standard deviation (range). Female Male Holotype Paratypes Paratypes n L ± 35.2 ( ) 447 ± 34 ( ) a ± 1.95 (25-32) 28.7 ± 2.2 (23-31) b ± 0.7 ( ) 6.2 ± 0.6 ( ) b ± 0.45 ( ) 4.1 ± 0.4 ( ) c ± 1.2 (13-17) 15 ± 1.3 (12-17) c ± 0.2 ( ) 2.6 ± 0.3 (2-3.1) V 76 78± 1.1 (76-79) Maximum body diam ± 1.3 (15-20) 16 ± 0.7 (14-17) Body diam. at vulva ± 1.0 (15-18) Body diam. at anus ± 0.8 (10-12) 11.3 ± 0.6 (10-12) Head diam ± 0.4 (8-9) 7.3 ± 0.3 (7-8) Head height ± 0.4 (2-3.2) 2.5 ± 0.2 ( ) Stylet length ± 0.4 (16-18) 14.6 ± 0.5 (14-15) Stylet knob width ± 0.3 (5-6) 3.8 ± 0.4 (3-4.5) Stylet knob height ± 0.4 (2-3) 1.7 ± 0.4 ( ) DGO ± 0.4 ( ) 2.4 ± 0.4 ( ) Anterior end to metacorpus ± 5.8 (35-57) 50 ± 2.3 (46-56) to secretory/excretory pore ± 6.2 (63-88) 75 ± 6.9 (63-87) to pharyngo-intestinal junction ± 8.0 (56-86) 72 ± 4.1 (63-78) to posterior end pharyngeal glands ± 10.5 ( ) 109 ± 7.2 (99-120) to vulva ± 27.4 ( ) Pharyngeal gland length ± 7.7 (32-54) 36 ± 5.9 (32-48) Length posterior uterine sac ± 3.3 (12-25) Lateral field width ± 0.5 (5-7) 4.4 ± 0.5 (4-5) Tail length ± 2.6 (30-38) 29.9 ± 3.0 (23-35) Tail annules ± 2.8 (31-39) Spicule length 15.1 ± 0.8 ( ) Gubernaculum length 5.0 ± 0.5 (4.5-6) Testis 194 ± 29.3 ( ) Phasmid to tail tip ± 1.6 (13-19) 13.5 ± 2.0 ( ) Hyaline part ± 0.5 ( ) Head diam./height ± 0.5 ( ) 2.9 ± 0.2 ( ) Stylet diam./height ± 0.4 ( ) 2.3 ± 0.5 ( ) Fig. 2. Pratylenchus dunensis sp. n. A: Female tail terminus; B: Female tail terminus with indentations next to tail tip; C, D: Female lateral field at mid-body and areolation (arrows). (Scale bar = 10 µm.) Vol. 8(1),

6 E. de la Peña et al. Fig. 3. Pratylenchus dunensis sp. n. SEM studies. A: Lateral view of female head region; B: Lateral view of male head region; C: En face view of female lip region; D: Female lateral field view at mid-body; E: Excretory/secretory pore; F: Lateral field and phasmid (arrow) on female tail. 84 Nematology

7 A new species of Pratylenchus from dune grass Fig. 4. Restriction fragments of amplified internal transcribed spacers of Pratylenchus dunensis sp. n., P. penetrans and P. brzeskii. A: P. penetrans; B:P. brzeskii; C:P. dunensis sp.n.(x = DNA ladder, 100 bp, Promega.). Table 2. Pairwise D2D3 sequence alignment similarity (%) of Pratylenchus dunensis sp. n., P. brzeskii and P. penetrans. % Identity Pratylenchus P. brzeskii P. penetrans dunensis sp. n. P. dunensis sp. n P. brzeskii P. penetrans 100 between outer incisures, lateral field partially areolated through whole body, rounded to oval spermatheca filled with round sperm, conical tail with annules, tail tip rounded and smooth with short, conspicuous, hyaline part (ca 2 µm), phasmid located between inner lateral field incisures in posterior half of tail. Pratylenchus dunensis sp. n. is further characterised by the abundant presence of males (nearly 50% of adults). As is the case for P. dunensis sp. n. the following species all have two lip annules and a non-crenate tail: P. acuticaudatus Braasch & Dekker, 1989; P. agilis Thorne & Malek, 1968; P. alleni Ferris, 1961; P. angulatus Siddiqi, 1994; P. brachyurus (Godfrey, 1929) Filipjev & Schuurmans Stekhoven, 1941; P. brzeskii Karssen, Waeyenberge & Moens, 2000; P. coffeae (Zimmermann, 1898) Filipjev & Schuurmans Stekhoven, 1941; P. hexincisus Taylor & Jenkins, 1957; P. jordanensis Hashim, 1984; P. loosi Loof, 1960; P. macrostylus Wu, 1971; P. neglectus (Rensch, 1924) Filipjev & Schuurmans Stekhoven, 1941; P. neobrachyurus Siddiqi, 1994; P. pseudocoffeae Mizukubo, 1992; P. scribneri Steiner in Sherbakoff & Stanley, 1943; and P. silvaticus Brzeski, Morphologically, P. acuticaudatus, P. agilis, P. angulatus, P. brachyurus, P. hexincisus, P. jordanensis, P. macrostylus, P. neglectus and P. scribneri differ from P. dunensis sp. n. in having an empty spermatheca (males absent or very rare). Pratylenchus alleni differs by a smaller stylet (<15 vs µm) and a bluntly rounded tail with phasmids in the anterior half of the tail (vs conical tail with phasmids posterior to mid-tail). Pratylenchus coffeae and P. pseudocoffeae differ in a generally more posterior vulva position (V = vs 76-79), a truncate tail (vs conical), elongated spermatheca (vs round to oval) and a well differentiated post uterine branch (vs undifferentiated and short). Pratylenchus loosi differs from P. dunensis sp. n. in having a more posterior vulva position (V = vs 76-79), a broad lateral field with incisures, and a relatively shorter ratio tail (c = vs 13-17). Pratylenchus neobrachyurus is shorter ( vs µm) and broader (a = vs 25-32) with a more posterior vulva (V = vs 76-79), fewer tail Vol. 8(1),

8 E. de la Peña et al. Table 3. Length (bp) of restriction fragments of rdna Internal Transcribed Spacer regions for Pratylenchus dunensis sp. n., P. brzeskii and P. penetrans. Enzyme Pratylenchus dunensis sp. n. P. penetrans P. brzeskii CfoI 415, , , 70 EcoRI No restriction 405, 295 No restriction HindIII 400, , 290 No restriction MspI No restriction No restriction 425, 255 PstI 400, , , 300 annules (19-23 vs 31-39) and a non-areolated lateral field. Pratylenchus silvaticus differs from Pratylenchus dunensis sp. n. in head morphology (head not set-off), a compact pharyngeal lobe (vs relatively long) and tail tapering before terminus. The ratio between body length and greatest body diameter is also different between the two species (a = vs 25-31). The vulva position in P. silvaticus is also slightly more posterior than in P. dunensis sp. n. (V = vs 76-79). Pratylenchus brzeskii shares the same habitat with P. dunensis sp. n. However, P. brzeskii is longer than P. dunensis sp. n. ( vs µm), has a greater body diameter (23-25 vs µm), longer pharyngeal glands ( vs µm), a longer stylet (18-19 vs µm) and tail length (41-51 vs µm). Another difference is found in the lateral field. In both species the lateral field at mid-body forms three ridges, the middle ridge being narrower than the outer ones. However, in P. brzeskii the inner ridge is considerably narrower than in P. dunensis sp. n. (ridge occupying vs 60-80% of the distance between the outer lines). The most common morphotype of P. penetrans has three lip annuli. However, some populations of P. penetrans have been reported with two lip annuli (Tarté & Mai, 1976). Compared with P. dunensis sp. n., P. penetrans is generally more slender (a = vs ) and shows a larger range in the pharyngeal gland/body length ratio (b = vs ). Some tail characteristics are further useful characters to distinguish the two species: the tail of P. penetrans is vs µm long in P. dunensis sp. n., and there are more tail annules in P. dunensis sp. n. than in P. penetrans (31-39 vs 15-27). The lateral field is a further useful character to separate the species (P. penetrans with four equidistant incisures vs four non-equidistant incisures in P. dunensis sp. n.). Within the genus Pratylenchus, three species are described as having two lip annules and a crenate tail, viz P. flakkensis Seinhorst, 1968, P. estoniensis Ryss, 1982 and P. gibbicaudatus Minawa & Nozuma, These species show an obvious crenation of the tail whereas P. dunensis sp. n. has a smooth tail tip. Pratylenchus flakkensis also has an elongated spermatheca (vs round to oval), a smaller number of tail annules (14-26 vs 31-39) and males are uncommon. Pratylenchus estoniensis has six lateral lines (vs four) and an empty spermatheca (vs filled with round sperm) and P. gibbicaudatus has an empty spermatheca and males absent or rarely present. MOLECULAR CHARACTERISATION The D2D3 LSU sequences revealed clear differences between P. dunensis sp. n. and P. brzeskii and P. penetrans, the two Pratylenchus species with which the new species might be confused (Table 2). Pratylenchus penetrans is genetically closer to P. dunensis sp. n. than P. brzeskii. The size of the ITS-PCR product was different for the three species. For P. dunensis sp. n. and P. penetrans a product of nearly 700 bp (695 and 705 bp, respectively) was obtained, whereas for P. brzeskii the PCR yielded a slightly smaller fragment (680 bp). The ITS-RFLP patterns obtained after the restriction of the ITS-region allow the three species to be differentiated (Table 3, Fig. 4, bottom panel). Pratylenchus brzeskii can be separated from the other two species by CfoI, HindIII, MspI and PstI. Pratylenchus penetrans can be separated from the two other species by EcoRI. Pratylenchus dunensis sp. n. cannot be separated with a single digestion, but can be distinguished by comparing the RFLP patterns obtained with EcoRI and MspI; EcoRI only digested the PCR product of P. penetrans and MspI only yielded restriction fragments for P. brzeskii. Discussion The population described in this paper as P. dunensis sp. n. was originally identified as P. penetrans. However, further studies of additional molecular data, morphological characters and morphometrics indicated that 86 Nematology

9 A new species of Pratylenchus from dune grass the differences compared to P. penetrans justified recognition at the species level. In addition to the morphology and morphometrics, the sequence comparison of the D2D3 rdna expansion region clearly separates the new species from both P. brzeskii and P. penetrans. We found at least 25% and 11% nucleotide difference with respect to P. brzeskii and P. penetrans, respectively. This rdna region has been widely used to separate different groups of nematodes at species level, including pratylenchids (Duncan et al., 1999; Handoo et al., 2001). The difference between P. dunensis sp. n. and P. penetrans is further confirmed by differences in patterns obtained after endonuclease restriction of the ITS regions. The RFLP profiles we obtained with CfoI, HindIII and MspI were the same as those obtained by Karssen et al. (2001); however, EcoRI yielded a different pattern for both species (no restriction vs two bands) and clearly separates the species. It is clear that the number of restriction enzymes used in the first observations was not sufficient for species discrimination, the variability of DNA fragments being better estimated by observing their sequences rather than restriction patterns. As a consequence, our results refute the previously reported presence of P. penetrans parasitising A. arenaria in The Netherlands and Seinhorst s (1968) hypothesis that P. penetrans is not native to The Netherlands therefore remains valid. The distribution of P. dunensis sp. n. in coastal fore dunes with vigorous A. arenaria and E. farctus suggests an adaptation of the species to this habitat and thus might play a role in the dynamics of the plant community in coastal dunes. Pratylenchus dunensis sp. n. shares the habitat with other migratory or sedentary parasitic nematodes specific for A. arenaria such as P. brzeskii, Meloidogyne maritima (Jepson, 1987) Karssen, Van Aelst & Cook, 1998, M. duytsi Karssen, Van Aelst & Van der Putten, 1998, and Heterodera arenaria Cooper, 1955 (see Karssen et al., 1998a, b). Recent studies have shown that Pratylenchus spp. and H. arenaria colonise newly deposited sand layers, thereby indicating that they follow root-growth through the different seasons (Van der Stoel et al., 2002). These authors proved the negative influence of the nematodes on the growth of A. arenaria. Therelationships between the different groups of nematodes and the host plant are not yet clear. However, glasshouse trials showed that, on A. arenaria, Pratylenchus species are competing for food with H. arenaria and M. maritima (Brinkman, 2004). To have a clear picture of the ecological processes involving plant-parasitic nematodes in coastal dunes the effect of P. dunensis sp. n. should, therefore, be considered. Acknowledgements We thank Roel Wagenaar, Henk Duyts and Prof. Wim van der Putten at The Netherlands Institute of Ecology (NIOO), Heteren, The Netherlands, for helping in sampling at Groote Keeten. This work was funded by EU project EcoTrain: RTN References BRINKMAN, P. (2004). Interactions among endoparasitic rootfeeding nematodes; consequences for nematodes and host plant. PhD Thesis, Wageningen University, Wageningen, The Netherlands, 96 pp. BRZESKI, M.W. (1998). Nematodes of Tylenchina in Poland and temperate Europe. Warsaw, Poland, Museum and Institute of Zoology, Polish Academy of Sciences, 398 pp. COURTNEY, W.D., POLLEY, D.&MILLER, V.I. (1955). TAF an improved fixative in nematode technique. Plant Disease Reporter 39, DE LEY, P.,FELIX, M.A., FRISSE, L.M., NADLER, S.A., STERNBERG, P.W. & THOMAS, W.K. (1999). Molecular and morphological characterisation of two reproductively isolated species with mirror image anatomy (Nematoda: Cephalobidae). Nematology 6, DE ROOIJ-VAN DER GOES, P.C.E.M, VAN DER PUTTEN, W.H. & VAN DIJK, C. (1995). Analysis of nematodes and soil-borne fungi from Ammophila arenaria (marram grass) in Dutch coastal foredunes by multivariate techniques. European Journal of Plant Pathology 101, DUNCAN, L.W., INSERRA, R.N., THOMAS, W.K., DUNN, D., MUSTIKA, I., FRISSE, L.M., MENDES, M.L., MORRIS, K. & KAPLAN, D.T. (1999). Molecular and morphological analysis of isolates of Pratylenchus coffeae and closely related species. Nematropica 29, HALL, T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Serial 41, HANDOO, Z.A., CARTA, L.K. & SKANTAR, A.M. (2001). Morphological and molecular characterisation of Pratylenchus arlingtoni n. sp., P. convallariae and P. fallax (Nematoda : Pratylenchidae). Nematology 3, HOOPER, D.J. & EVANS, K. (1993). Extraction, identification and control of plant parasitic nematodes. In: Evans, K., Trudgill, D.L. & Webster, J.M. (Eds). Plant parasitic nematodes in temperate agriculture. Wallingford, UK, CABI Publishing, pp KARSSEN, G., VAN AELST, A. & COOK, R. (1998a). Redescription of the root-knot nematode Meloidogyne maritima Vol. 8(1),

10 E. de la Peña et al. Jepson, 1987 (Nematoda: Heteroderidae), a parasite of Ammophila arenaria (L.) Link. Nematologica 44, KARSSEN, G.,VAN AELST, A.&VAN DER PUTTEN, W.H. (1998b). Meloidogyne duytsi sp. n. (Nematoda: Heteroderidae), a root-knot nematode from Dutch coastal foredunes. Fundamental and Applied Nematology 21, KARSSEN, G., WAEYENBERGE, L. & MOENS, M. (2000). Pratylenchus brzeskii sp. nov. (Nematoda: Pratylenchidae), a root-lesion nematode from European coastal dunes. Annales Zoologici 50, KARSSEN, G., VAN AELST, A., WAEYENBERGE, L. & MOENS, M. (2001). Observations on Pratylenchus penetrans Cobb, 1917 parasitizing the coastal dune grass Ammophila arenaria (L.) Link in The Netherlands. Journal of Nematode Morphology and Systematics 4, 1-9. SEINHORST, J.W. (1950). De betekenis van de toestand van de grond voor het optreden van aantasting door het stengelaaltje (Ditylenchus dipsaci Kühn). Tijdschrift voor Plantenziekten 10, SEINHORST, J.W. (1968). Three new Pratylenchus species with a discussion of the structure of the cephalic framework and of the spermatheca in this genus. Nematologica 14, SELISKAR, D.M. & HUETTEL, R.N. (1993). Nematode involvement in the dieout of Ammophila breviligulata (Poaceae) on the Mid-Atlantic coastal dunes of the United States. Journal of Coastal Research 9, TARTÉ, R.& MAI, W.F. (1976). Morphological variation in Pratylenchus penetrans. Journal of Nematology 8, THOMPSON, J., HIGGINS, D. & GIBSON, T. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, VAN DER PUTTEN, W.H.& PETERS, B.A. (1997). How soilborne pathogens may affect plant competition. Ecology 78, VAN DER STOEL, C.D., VAN DER PUTTEN, W.H.&DUYTS, H. (2002). Development of a negative plant-soil feedback in the expansion zone of the clonal grass Ammophila arenaria following root formation and nematode colonization. Journal of Ecology 90, VERSCHOOR, B.C., PRONK, T.E., DE GOEDE, R.G.M. & BRUSSAARD, L. (2002). Could plant-feeding nematodes affect the competition between grass species during succession in grasslands under restoration management? Journal of Ecology 90, WAEYENBERGE, L., RYSS, A., MOENS, M., PINOCHET, J. & VRAIN, T.C. (2000). Molecular characterisation of 18 Pratylenchus species using rdna Restriction Fragment Length Polymorphism. Nematology 2, Nematology