Nematology, 2006, Vol. 8(1), 79-88 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 VA N 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 (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. Keywords – D2D3, description, Elymus farctus, ITS-rDNA, molecular, morphology, morphometrics RFLP. Plant-parasitic nematodes and their influence on plant plants in coastal dunes (Seliskar & Huettel, 1993; Karssen competition in semi-natural and natural systems such as et al., 2000). coastal dunes have received considerable attention in the In 1998, specimens of the genus Pratylenchus were de- past few years (De Rooij-Van der Goes et al., 1995; tected in the roots and rhizosphere of A. arenaria sam- Van der Putten & Peters, 1997; Verschoor et al., 2002). pled in the dune area of Groote Keeten, province Noord- Two species of the genus Pratylenchus Filipjev, 1936, Holland, The Netherlands. Although the population dif- viz P. brzeskii Karssen, Waeyenberge & Moens, 2000 fered from P. penetrans by the number of lip annules, tail and P. penetrans (Cobb, 1917) Filipjev & Schuurmans morphology and some morphometrics, it was identified Stekhoven, 1941, have been reported on Ammophila as P. penetrans because the ITS-RFLP of the population arenaria (L.) Link and A. breviligulata Fern., respectively, were identical to those of P. penetrans as published by and are thought to be involved in the die-out of these Waeyenberge et al. (2000). As a result, P. penetrans was ∗ Corresponding author, e-mail: [email protected] © Koninklijke Brill NV, Leiden, 2006 79 Also available online - www.brill.nl E. de la Peña et al. reported in coastal dunes of The Netherlands for the first MOLECULAR STUDIES time (Karssen et al., 2001). During 2002 and 2003, nematode surveys were made in For each of the nematode species, DNA was extracted dune areas with vigorous A. arenaria stands along the Eu- from previously identified female nematodes. Each ne- ropean Atlantic coast. Once again, specimens similar to P. matode was placed in 25 µl digestion buffer (100 mM penetrans but showing the above mentioned deviations in morphology and morphometrics were detected at different KCl, 20 mM Tris pH 8, 3 mM MgCl2, 2 mM DTT, 0.9% sampling sites. Their D2D3 LSU sequences showed fur- Tween 20) diluted with 25 µl water and proteinase K (600 µg/ml). The extract was incubated at 65◦Cfor2h ther differences from those of P. penetrans populations. ◦ As a consequence the identity of the population from and then 5 min at 95 C to denaturise proteinase K. The ◦ Groote Keeten was reconsidered and DNA sequence com- crude extract was kept at −70 C until use. Five µlofthe parisons with more P.penetrans populations indicated that crude extract was used for PCR amplification. Primers and the differences were too striking to be taken as intra- PCR conditions for both the D2D3 and the ITS (including specific variation. Therefore, more comparisons of D2D3 the 5.8S gene and flanking areas of the 18S and 28S genes sequences and eventually also of ITS-RFLP, as made pre- of rDNA) amplification were as described by De Ley et al. viously, were combined with extra observations on the (1999) and Waeyenberge et al. (2000), respectively. Af- morphology and morphometrics. The additional data re- ter electrophoresis in 1% TAE-buffered agarose gels (1 h, vealed that the population belongs to a new species, which 100 V), the PCR product was visualised under UV light. in this paper is described as Pratylenchus dunensis sp. n. 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 Effi- Materials and methods ciency Competent Cells (Promega, Leiden, The Nether- lands). Several clones were isolated using blue/white NEMATODE POPULATIONS selection and sequenced using vector primers with a BigDye Terminator Cycle Sequencing Ready Reaction Pratylenchus dunensis sp. n. was isolated from soil and Kit (PE Applied Biosystems, Foster City, CA, USA). roots collected in the rhizosphere of A. arenaria in the The resulting products were purified using a Centri- coastal dunes of Groote Keeten (Province Noord-Holland, flex Gel Filtration Cartridge (Edge Biosystems, Gaithers- The Netherlands). The new species was compared with a burg, MD, USA). DNA samples were sequenced by ABI population of P. penetrans isolated from Wisteria sinen- Prism 377 DNA Sequencer (PE Applied Biosystems). sis Sweet in Maaseik (Belgium) and a population of DNA sequences were edited manually with Chromas P. brzeskii collected on A. arenaria in Biarritz (France). 1.45 (Technelysium, Helensvale, Australia), and aligned Females and males were extracted from the roots in a mist chamber (Seinhorst, 1950). with Clustal X 1.64 (default options) (Thompson et al., 1997). The sequences of Pratylenchus dunensis sp.n.arede- MORPHOLOGICAL STUDY posited in EMBL (http://www.embl-heidelberg.de/) under the accession numbers AJ890459, AJ890460, AJ890461 For light microscopical observations, males and fe- and AJ890462; the P. brzeskii sequence was deposited un- males were fixed in TAF (Courtney et al., 1955). Ne- matodes were mounted in anhydrous glycerine using the der the accession number AJ890463. slow method of Hooper and Evans (1993). Morphomet- Differences between sequences were estimated using rics were taken using an Olympus BX50 compound mi- the DNA distance option provided by BioEdit sequence croscope equipped with Leica image-capture IM500 sys- alignment editor (Hall, 1999). tem and software. Amplified PCR products were purified using a Qiagen For scanning electron microscopy (SEM), fixed nema- Gel Purification Kit. After purification, one µl of puri- todes were coated with 45 nm of platinum in a prepa- fied PCR product was digested with one of five restric- ration chamber (CT-1500HT, Oxford Instruments, High tion enzymes, viz., CfoI, HindIII, PstI, EcoRI and MspI, Wycombe, UK) and observed with a Jeol 6300F field according to the instructions of the manufacturer (Amer- emission electron microscope at 3.5 kV. sham Bioscience, Little Chalfont, UK). 80 Nematology A new species of Pratylenchus from dune grass Pratylenchus dunensis* sp. n. Male = P. penetrans apud Karssen et al., 2001 Occurring abundantly (nearly 50% of adults). Morpho- (Figs 1-4) logically similar to females, but smaller for all non-sexual characters. Head characters as in females, but more trun- MEASUREMENTS cated in outline. Apical annules slightly raised. Incom- plete transverse incisures and variation in lip annuli as de- scribed for females. Single testis anteriorly outstretched. See Table 1. Spicules and gubernaculum ventrally curved. Bursa en- closing tail. Ventral side of bursa coarsely crenate; phas- DESCRIPTION midial orifice on bursa located at almost mid distance be- tween cloaca and tail tip. Female TYPE HOST AND LOCALITY Body vermiform, slender and tapering towards both ends. Cuticle finely annulated with annules 1-1.3 µm Pratylenchus dunensis sp. n. was found in roots of Am- wide at mid-body. Head slightly set-off, three times height mophila arenaria growing in the fore dunes of Groote in diam., rounded with prominent cephalic sclerotisation Keeten, Province Noord Holland, The Netherlands. The and vestibule extension. Two lip annuli, often with one species was also detected in the roots of Elymus farctus or two incomplete, transverse incisures not visible with Viv. present at the same area. The distribution of P. dunen- light microscope. Labial disc oval, elevated, fused with sis sp. n. seems to be favoured by conditions in front dunes four submedial lips forming smooth, dumb-bell shaped, where there is considerable sand deposition and the two headcap. Cephalic sensilla marked by subtle depression dune grasses grow vigorously.