Description of Pratylenchus dunensis sp n (NematodaPratylenchidae) a root-lesion nematode associated with the dune
grass Ammophila arenaria (L) Link
Eduardo DE LA PENtildeA 1lowast Maurice MOENS 12 Adriaan VAN AELST 3 and Gerrit KARSSEN 45
1 Agricultural Research Centre Crop Protection Department Burg van Gansberghelaan 96 9820Merelbeke Belgium
2 Gent University Laboratory for Agrozoology Coupure 653 9000 Gent Belgium3 Wageningen University amp Research Centre Laboratory of Plant Cell Biology Arboretumlaan 4
6703 BD Wageningen The Netherlands4 Plant Protection Service Nematology Section PO Box 9102 6700 HC Wageningen The Netherlands
5 Wageningen University amp Research Centre Laboratory of Nematology Binnenhaven 56709 PD Wageningen The Netherlands
Received 4 April 2005 revised 7 November 2005Accepted for publication 7 November 2005
Summary ndash A root-lesion nematode Pratylenchus dunensis sp n is described and illustrated from Ammophila arenaria (L) Linka grass occurring abundantly in coastal dunes of Atlantic Europe The new species is characterised by medium sized (454-579 microm)slender vermiform females and males having two lip annuli (sometimes three to four incomplete incisures only visible with scanningelectron microscopy) medium to robust stylet (ca 16 microm) with robust stylet knobs slightly set off long pharyngeal glands (ca 42 microm)lateral field with four parallel non-equidistant lines the middle ridge being narrower than the outer ones lateral field with partialareolation and lines converging posterior to the phasmid which is located between the two inner lines of the lateral field in the posteriorhalf of the tail round spermatheca filled with round sperm vulva at 78 of total body length and with protruding vulval lips posterioruterine sac relatively short (ca 19 microm) cylindrical tail (ca 33 microm) narrowing in the posterior third with smooth tail tip and withconspicuous hyaline part (ca 2 microm) Males occur abundantly and present similar characteristics except for smaller dimensions for allmorphological characters but the head region is more truncated in outline than the female spicule length is ca 15 microm and testis length isca 195 microm Nucleotide sequences of the rDNA expansion region D2D3 differed from the morphologically similar species P penetransand P brzeskii that also occur in coastal dunes These differences are supported by PCR-RFLP of the ITS-rDNA Pratylenchus dunensissp n was also found parasitising roots of Elymus farctus Viv
Plant-parasitic nematodes and their influence on plantcompetition in semi-natural and natural systems such ascoastal dunes have received considerable attention in thepast few years (De Rooij-Van der Goes et al 1995Van der Putten amp Peters 1997 Verschoor et al 2002)Two species of the genus Pratylenchus Filipjev 1936viz P brzeskii Karssen Waeyenberge amp Moens 2000and P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 have been reported on Ammophilaarenaria (L) Link and A breviligulata Fern respectivelyand are thought to be involved in the die-out of these
plants in coastal dunes (Seliskar amp Huettel 1993 Karssenet al 2000)
In 1998 specimens of the genus Pratylenchus were de-tected in the roots and rhizosphere of A arenaria sam-pled in the dune area of Groote Keeten province Noord-Holland The Netherlands Although the population dif-fered from P penetrans by the number of lip annules tailmorphology and some morphometrics it was identifiedas P penetrans because the ITS-RFLP of the populationwere identical to those of P penetrans as published byWaeyenberge et al (2000) As a result P penetrans was
reported in coastal dunes of The Netherlands for the firsttime (Karssen et al 2001)
During 2002 and 2003 nematode surveys were made indune areas with vigorous A arenaria stands along the Eu-ropean Atlantic coast Once again specimens similar to Ppenetrans but showing the above mentioned deviations inmorphology and morphometrics were detected at differentsampling sites Their D2D3 LSU sequences showed fur-ther differences from those of P penetrans populationsAs a consequence the identity of the population fromGroote Keeten was reconsidered and DNA sequence com-parisons with more P penetrans populations indicated thatthe differences were too striking to be taken as intra-specific variation Therefore more comparisons of D2D3sequences and eventually also of ITS-RFLP as made pre-viously were combined with extra observations on themorphology and morphometrics The additional data re-vealed that the population belongs to a new species whichin this paper is described as Pratylenchus dunensis sp n
Materials and methods
NEMATODE POPULATIONS
Pratylenchus dunensis sp n was isolated from soil androots collected in the rhizosphere of A arenaria in thecoastal dunes of Groote Keeten (Province Noord-HollandThe Netherlands) The new species was compared with apopulation of P penetrans isolated from Wisteria sinen-sis Sweet in Maaseik (Belgium) and a population ofP brzeskii collected on A arenaria in Biarritz (France)Females and males were extracted from the roots in a mistchamber (Seinhorst 1950)
MORPHOLOGICAL STUDY
For light microscopical observations males and fe-males were fixed in TAF (Courtney et al 1955) Ne-matodes were mounted in anhydrous glycerine using theslow method of Hooper and Evans (1993) Morphomet-rics were taken using an Olympus BX50 compound mi-croscope equipped with Leica image-capture IM500 sys-tem and software
For scanning electron microscopy (SEM) fixed nema-todes were coated with 45 nm of platinum in a prepa-ration chamber (CT-1500HT Oxford Instruments HighWycombe UK) and observed with a Jeol 6300F fieldemission electron microscope at 35 kV
MOLECULAR STUDIES
For each of the nematode species DNA was extractedfrom previously identified female nematodes Each ne-matode was placed in 25 microl digestion buffer (100 mMKCl 20 mM Tris pH 8 3 mM MgCl2 2 mM DTT 09Tween 20) diluted with 25 microl water and proteinase K(600 microgml) The extract was incubated at 65C for 2 hand then 5 min at 95C to denaturise proteinase K Thecrude extract was kept at minus70C until use Five microl of thecrude extract was used for PCR amplification Primers andPCR conditions for both the D2D3 and the ITS (includingthe 58S gene and flanking areas of the 18S and 28S genesof rDNA) amplification were as described by De Ley et al(1999) and Waeyenberge et al (2000) respectively Af-ter electrophoresis in 1 TAE-buffered agarose gels (1 h100 V) the PCR product was visualised under UV light
DNA was excised from the gels using the QIAquick GelExtraction Kit (Qiagen Hilden Germany) cloned intopGEM-T vector and transformed into JM109 High Effi-ciency Competent Cells (Promega Leiden The Nether-lands) Several clones were isolated using bluewhiteselection and sequenced using vector primers with aBigDye Terminator Cycle Sequencing Ready ReactionKit (PE Applied Biosystems Foster City CA USA)The resulting products were purified using a Centri-flex Gel Filtration Cartridge (Edge Biosystems Gaithers-burg MD USA) DNA samples were sequenced by ABIPrism 377 DNA Sequencer (PE Applied Biosystems)DNA sequences were edited manually with Chromas145 (Technelysium Helensvale Australia) and alignedwith Clustal X 164 (default options) (Thompson et al1997)
The sequences of Pratylenchus dunensis sp n are de-posited in EMBL (httpwwwembl-heidelbergde) underthe accession numbers AJ890459 AJ890460 AJ890461and AJ890462 the P brzeskii sequence was deposited un-der the accession number AJ890463
Differences between sequences were estimated usingthe DNA distance option provided by BioEdit sequencealignment editor (Hall 1999)
Amplified PCR products were purified using a QiagenGel Purification Kit After purification one microl of puri-fied PCR product was digested with one of five restric-tion enzymes viz CfoI HindIII PstI EcoRI and MspIaccording to the instructions of the manufacturer (Amer-sham Bioscience Little Chalfont UK)
80 Nematology
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 bothends Cuticle finely annulated with annules 1-13 micromwide at mid-body Head slightly set-off three times heightin diam rounded with prominent cephalic sclerotisationand vestibule extension Two lip annuli often with oneor two incomplete transverse incisures not visible withlight microscope Labial disc oval elevated fused withfour submedial lips forming smooth dumb-bell shapedheadcap Cephalic sensilla marked by subtle depressionon each submedial lip SEM en face view showingoval prestoma Stylet finely shaped relatively short withrobust anteriorly indented set-off knobs Stylet conelength equal or shorter than length of shaft plus knobsHemizonid near isthmus level 2-3 microm in length anteriorto oval shaped secretory-excretory pore located betweennerve ring and pharyngeal junction Pharyngeal glandlobe three body diam long Vulva with well-developedlips often protruding Lateral field with four lateral linesstarting posterior to stylet occupying ca one-third ofbody diam at mid-body At pharyngeal-vulva regionmiddle ridge narrower than outer ones at mid-body widthof inner ridge 60-80 of distance between outer linesOuter incisures partially areolated with striae crossingouter lateral field ridges every 1-15 microm Lateral linesconverging posterior to phasmid Genital tract mono-prodelphic with single row of oocytes Spermatheca roundto slightly oval filled with rounded sperm Post-uterinesac undifferentiated short Tail cylindrical with distinctannulations narrowing in posterior third with smoothdome-shaped terminus some specimens (one in fiveof adult females) with one or two indentations next tosmooth tail tip Hyaline part distinct Anus round to ovalshaped Small rounded phasmid located between innerlateral field lines posterior to mid-tail
Specific epithet derived from the dune habitat
Male
Occurring abundantly (nearly 50 of adults) Morpho-logically similar to females but smaller for all non-sexualcharacters Head characters as in females but more trun-cated in outline Apical annules slightly raised Incom-plete transverse incisures and variation in lip annuli as de-scribed for females Single testis anteriorly outstretchedSpicules and gubernaculum ventrally curved Bursa en-closing tail Ventral side of bursa coarsely crenate phas-midial orifice on bursa located at almost mid distance be-tween cloaca and tail tip
TYPE HOST AND LOCALITY
Pratylenchus dunensis sp n was found in roots of Am-mophila arenaria growing in the fore dunes of GrooteKeeten Province Noord Holland The Netherlands Thespecies was also detected in the roots of Elymus farctusViv present at the same area The distribution of P dunen-sis sp n seems to be favoured by conditions in front duneswhere there is considerable sand deposition and the twodune grasses grow vigorously In older more stabiliseddunes this species is less abundant
OTHER LOCALITIES AND HOSTS
Pratylenchus dunensis sp n has also been found inOostvoorne The Netherlands parasitising Ammophilaarenaria and Elymus farctus roots and in fore dunes atBlakeney Point Norfolk UK on A arenaria
TYPE MATERIAL
One holotype female six female and seven maleparatypes at the Nematode Collection of Wageningen(collection numbers WT3399 and WT3400) Univer-sity and Research Centre Wageningen The NetherlandsEight male and eight female paratypes at the Museumvoor Dierkunde (collection numbers UGMD 104068 andUGMD 104069) University of Gent KL Ledeganck-straat 35 9000 Gent Belgium
DIAGNOSIS AND RELATIONSHIPS
Pratylenchus dunensis sp n is characterised by twolip annuli with incomplete transverse incisures a stylet(ca 16 microm) with robust anteriorly indented and set offknobs vulva at 78 of body length lateral field withfour parallel lines unequally spaced from pharynx tovulva the inner ridge occupying 60-80 of the distance
Vol 8(1) 2006 81
E de la Pentildea et al
Fig 1 Pratylenchus dunensis sp n A Entire female body lateral view B Entire male body lateral view C Male cephalic regionD Female cephalic region E Female anterior end including pharyngeal gland F Female vulval region ovary and spermathecaG Lateral field at mid-body H Female tail tips I Female posterior end J Male posterior end
82 Nematology
A new species of Pratylenchus from dune grass
Table 1 Morphometric characters of Pratylenchus dunensis n sp Measurements are in microm and in the form mean plusmn standard deviation(range)
Fig 2 Pratylenchus dunensis sp n A Female tail terminus B Female tail terminus with indentations next to tail tip C D Femalelateral field at mid-body and areolation (arrows) (Scale bar = 10 microm)
Vol 8(1) 2006 83
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Fig 3 Pratylenchus dunensis sp n SEM studies A Lateral view of female head region B Lateral view of male head region C Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
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E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
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E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
E de la Pentildea et al
reported in coastal dunes of The Netherlands for the firsttime (Karssen et al 2001)
During 2002 and 2003 nematode surveys were made indune areas with vigorous A arenaria stands along the Eu-ropean Atlantic coast Once again specimens similar to Ppenetrans but showing the above mentioned deviations inmorphology and morphometrics were detected at differentsampling sites Their D2D3 LSU sequences showed fur-ther differences from those of P penetrans populationsAs a consequence the identity of the population fromGroote Keeten was reconsidered and DNA sequence com-parisons with more P penetrans populations indicated thatthe differences were too striking to be taken as intra-specific variation Therefore more comparisons of D2D3sequences and eventually also of ITS-RFLP as made pre-viously were combined with extra observations on themorphology and morphometrics The additional data re-vealed that the population belongs to a new species whichin this paper is described as Pratylenchus dunensis sp n
Materials and methods
NEMATODE POPULATIONS
Pratylenchus dunensis sp n was isolated from soil androots collected in the rhizosphere of A arenaria in thecoastal dunes of Groote Keeten (Province Noord-HollandThe Netherlands) The new species was compared with apopulation of P penetrans isolated from Wisteria sinen-sis Sweet in Maaseik (Belgium) and a population ofP brzeskii collected on A arenaria in Biarritz (France)Females and males were extracted from the roots in a mistchamber (Seinhorst 1950)
MORPHOLOGICAL STUDY
For light microscopical observations males and fe-males were fixed in TAF (Courtney et al 1955) Ne-matodes were mounted in anhydrous glycerine using theslow method of Hooper and Evans (1993) Morphomet-rics were taken using an Olympus BX50 compound mi-croscope equipped with Leica image-capture IM500 sys-tem and software
For scanning electron microscopy (SEM) fixed nema-todes were coated with 45 nm of platinum in a prepa-ration chamber (CT-1500HT Oxford Instruments HighWycombe UK) and observed with a Jeol 6300F fieldemission electron microscope at 35 kV
MOLECULAR STUDIES
For each of the nematode species DNA was extractedfrom previously identified female nematodes Each ne-matode was placed in 25 microl digestion buffer (100 mMKCl 20 mM Tris pH 8 3 mM MgCl2 2 mM DTT 09Tween 20) diluted with 25 microl water and proteinase K(600 microgml) The extract was incubated at 65C for 2 hand then 5 min at 95C to denaturise proteinase K Thecrude extract was kept at minus70C until use Five microl of thecrude extract was used for PCR amplification Primers andPCR conditions for both the D2D3 and the ITS (includingthe 58S gene and flanking areas of the 18S and 28S genesof rDNA) amplification were as described by De Ley et al(1999) and Waeyenberge et al (2000) respectively Af-ter electrophoresis in 1 TAE-buffered agarose gels (1 h100 V) the PCR product was visualised under UV light
DNA was excised from the gels using the QIAquick GelExtraction Kit (Qiagen Hilden Germany) cloned intopGEM-T vector and transformed into JM109 High Effi-ciency Competent Cells (Promega Leiden The Nether-lands) Several clones were isolated using bluewhiteselection and sequenced using vector primers with aBigDye Terminator Cycle Sequencing Ready ReactionKit (PE Applied Biosystems Foster City CA USA)The resulting products were purified using a Centri-flex Gel Filtration Cartridge (Edge Biosystems Gaithers-burg MD USA) DNA samples were sequenced by ABIPrism 377 DNA Sequencer (PE Applied Biosystems)DNA sequences were edited manually with Chromas145 (Technelysium Helensvale Australia) and alignedwith Clustal X 164 (default options) (Thompson et al1997)
The sequences of Pratylenchus dunensis sp n are de-posited in EMBL (httpwwwembl-heidelbergde) underthe accession numbers AJ890459 AJ890460 AJ890461and AJ890462 the P brzeskii sequence was deposited un-der the accession number AJ890463
Differences between sequences were estimated usingthe DNA distance option provided by BioEdit sequencealignment editor (Hall 1999)
Amplified PCR products were purified using a QiagenGel Purification Kit After purification one microl of puri-fied PCR product was digested with one of five restric-tion enzymes viz CfoI HindIII PstI EcoRI and MspIaccording to the instructions of the manufacturer (Amer-sham Bioscience Little Chalfont UK)
80 Nematology
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 bothends Cuticle finely annulated with annules 1-13 micromwide at mid-body Head slightly set-off three times heightin diam rounded with prominent cephalic sclerotisationand vestibule extension Two lip annuli often with oneor two incomplete transverse incisures not visible withlight microscope Labial disc oval elevated fused withfour submedial lips forming smooth dumb-bell shapedheadcap Cephalic sensilla marked by subtle depressionon each submedial lip SEM en face view showingoval prestoma Stylet finely shaped relatively short withrobust anteriorly indented set-off knobs Stylet conelength equal or shorter than length of shaft plus knobsHemizonid near isthmus level 2-3 microm in length anteriorto oval shaped secretory-excretory pore located betweennerve ring and pharyngeal junction Pharyngeal glandlobe three body diam long Vulva with well-developedlips often protruding Lateral field with four lateral linesstarting posterior to stylet occupying ca one-third ofbody diam at mid-body At pharyngeal-vulva regionmiddle ridge narrower than outer ones at mid-body widthof inner ridge 60-80 of distance between outer linesOuter incisures partially areolated with striae crossingouter lateral field ridges every 1-15 microm Lateral linesconverging posterior to phasmid Genital tract mono-prodelphic with single row of oocytes Spermatheca roundto slightly oval filled with rounded sperm Post-uterinesac undifferentiated short Tail cylindrical with distinctannulations narrowing in posterior third with smoothdome-shaped terminus some specimens (one in fiveof adult females) with one or two indentations next tosmooth tail tip Hyaline part distinct Anus round to ovalshaped Small rounded phasmid located between innerlateral field lines posterior to mid-tail
Specific epithet derived from the dune habitat
Male
Occurring abundantly (nearly 50 of adults) Morpho-logically similar to females but smaller for all non-sexualcharacters Head characters as in females but more trun-cated in outline Apical annules slightly raised Incom-plete transverse incisures and variation in lip annuli as de-scribed for females Single testis anteriorly outstretchedSpicules and gubernaculum ventrally curved Bursa en-closing tail Ventral side of bursa coarsely crenate phas-midial orifice on bursa located at almost mid distance be-tween cloaca and tail tip
TYPE HOST AND LOCALITY
Pratylenchus dunensis sp n was found in roots of Am-mophila arenaria growing in the fore dunes of GrooteKeeten Province Noord Holland The Netherlands Thespecies was also detected in the roots of Elymus farctusViv present at the same area The distribution of P dunen-sis sp n seems to be favoured by conditions in front duneswhere there is considerable sand deposition and the twodune grasses grow vigorously In older more stabiliseddunes this species is less abundant
OTHER LOCALITIES AND HOSTS
Pratylenchus dunensis sp n has also been found inOostvoorne The Netherlands parasitising Ammophilaarenaria and Elymus farctus roots and in fore dunes atBlakeney Point Norfolk UK on A arenaria
TYPE MATERIAL
One holotype female six female and seven maleparatypes at the Nematode Collection of Wageningen(collection numbers WT3399 and WT3400) Univer-sity and Research Centre Wageningen The NetherlandsEight male and eight female paratypes at the Museumvoor Dierkunde (collection numbers UGMD 104068 andUGMD 104069) University of Gent KL Ledeganck-straat 35 9000 Gent Belgium
DIAGNOSIS AND RELATIONSHIPS
Pratylenchus dunensis sp n is characterised by twolip annuli with incomplete transverse incisures a stylet(ca 16 microm) with robust anteriorly indented and set offknobs vulva at 78 of body length lateral field withfour parallel lines unequally spaced from pharynx tovulva the inner ridge occupying 60-80 of the distance
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Fig 1 Pratylenchus dunensis sp n A Entire female body lateral view B Entire male body lateral view C Male cephalic regionD Female cephalic region E Female anterior end including pharyngeal gland F Female vulval region ovary and spermathecaG Lateral field at mid-body H Female tail tips I Female posterior end J Male posterior end
82 Nematology
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Table 1 Morphometric characters of Pratylenchus dunensis n sp Measurements are in microm and in the form mean plusmn standard deviation(range)
Fig 2 Pratylenchus dunensis sp n A Female tail terminus B Female tail terminus with indentations next to tail tip C D Femalelateral field at mid-body and areolation (arrows) (Scale bar = 10 microm)
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Fig 3 Pratylenchus dunensis sp n SEM studies A Lateral view of female head region B Lateral view of male head region C Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
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Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
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 bothends Cuticle finely annulated with annules 1-13 micromwide at mid-body Head slightly set-off three times heightin diam rounded with prominent cephalic sclerotisationand vestibule extension Two lip annuli often with oneor two incomplete transverse incisures not visible withlight microscope Labial disc oval elevated fused withfour submedial lips forming smooth dumb-bell shapedheadcap Cephalic sensilla marked by subtle depressionon each submedial lip SEM en face view showingoval prestoma Stylet finely shaped relatively short withrobust anteriorly indented set-off knobs Stylet conelength equal or shorter than length of shaft plus knobsHemizonid near isthmus level 2-3 microm in length anteriorto oval shaped secretory-excretory pore located betweennerve ring and pharyngeal junction Pharyngeal glandlobe three body diam long Vulva with well-developedlips often protruding Lateral field with four lateral linesstarting posterior to stylet occupying ca one-third ofbody diam at mid-body At pharyngeal-vulva regionmiddle ridge narrower than outer ones at mid-body widthof inner ridge 60-80 of distance between outer linesOuter incisures partially areolated with striae crossingouter lateral field ridges every 1-15 microm Lateral linesconverging posterior to phasmid Genital tract mono-prodelphic with single row of oocytes Spermatheca roundto slightly oval filled with rounded sperm Post-uterinesac undifferentiated short Tail cylindrical with distinctannulations narrowing in posterior third with smoothdome-shaped terminus some specimens (one in fiveof adult females) with one or two indentations next tosmooth tail tip Hyaline part distinct Anus round to ovalshaped Small rounded phasmid located between innerlateral field lines posterior to mid-tail
Specific epithet derived from the dune habitat
Male
Occurring abundantly (nearly 50 of adults) Morpho-logically similar to females but smaller for all non-sexualcharacters Head characters as in females but more trun-cated in outline Apical annules slightly raised Incom-plete transverse incisures and variation in lip annuli as de-scribed for females Single testis anteriorly outstretchedSpicules and gubernaculum ventrally curved Bursa en-closing tail Ventral side of bursa coarsely crenate phas-midial orifice on bursa located at almost mid distance be-tween cloaca and tail tip
TYPE HOST AND LOCALITY
Pratylenchus dunensis sp n was found in roots of Am-mophila arenaria growing in the fore dunes of GrooteKeeten Province Noord Holland The Netherlands Thespecies was also detected in the roots of Elymus farctusViv present at the same area The distribution of P dunen-sis sp n seems to be favoured by conditions in front duneswhere there is considerable sand deposition and the twodune grasses grow vigorously In older more stabiliseddunes this species is less abundant
OTHER LOCALITIES AND HOSTS
Pratylenchus dunensis sp n has also been found inOostvoorne The Netherlands parasitising Ammophilaarenaria and Elymus farctus roots and in fore dunes atBlakeney Point Norfolk UK on A arenaria
TYPE MATERIAL
One holotype female six female and seven maleparatypes at the Nematode Collection of Wageningen(collection numbers WT3399 and WT3400) Univer-sity and Research Centre Wageningen The NetherlandsEight male and eight female paratypes at the Museumvoor Dierkunde (collection numbers UGMD 104068 andUGMD 104069) University of Gent KL Ledeganck-straat 35 9000 Gent Belgium
DIAGNOSIS AND RELATIONSHIPS
Pratylenchus dunensis sp n is characterised by twolip annuli with incomplete transverse incisures a stylet(ca 16 microm) with robust anteriorly indented and set offknobs vulva at 78 of body length lateral field withfour parallel lines unequally spaced from pharynx tovulva the inner ridge occupying 60-80 of the distance
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Fig 1 Pratylenchus dunensis sp n A Entire female body lateral view B Entire male body lateral view C Male cephalic regionD Female cephalic region E Female anterior end including pharyngeal gland F Female vulval region ovary and spermathecaG Lateral field at mid-body H Female tail tips I Female posterior end J Male posterior end
82 Nematology
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Table 1 Morphometric characters of Pratylenchus dunensis n sp Measurements are in microm and in the form mean plusmn standard deviation(range)
Fig 2 Pratylenchus dunensis sp n A Female tail terminus B Female tail terminus with indentations next to tail tip C D Femalelateral field at mid-body and areolation (arrows) (Scale bar = 10 microm)
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Fig 3 Pratylenchus dunensis sp n SEM studies A Lateral view of female head region B Lateral view of male head region C Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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Fig 4 Restriction fragments of amplified internal transcribed spacers of Pratylenchus dunensis sp n P penetrans and P brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
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Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
E de la Pentildea et al
Fig 1 Pratylenchus dunensis sp n A Entire female body lateral view B Entire male body lateral view C Male cephalic regionD Female cephalic region E Female anterior end including pharyngeal gland F Female vulval region ovary and spermathecaG Lateral field at mid-body H Female tail tips I Female posterior end J Male posterior end
82 Nematology
A new species of Pratylenchus from dune grass
Table 1 Morphometric characters of Pratylenchus dunensis n sp Measurements are in microm and in the form mean plusmn standard deviation(range)
Fig 2 Pratylenchus dunensis sp n A Female tail terminus B Female tail terminus with indentations next to tail tip C D Femalelateral field at mid-body and areolation (arrows) (Scale bar = 10 microm)
Vol 8(1) 2006 83
E de la Pentildea 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 Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
Vol 8(1) 2006 85
E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
A new species of Pratylenchus from dune grass
Table 1 Morphometric characters of Pratylenchus dunensis n sp Measurements are in microm and in the form mean plusmn standard deviation(range)
Fig 2 Pratylenchus dunensis sp n A Female tail terminus B Female tail terminus with indentations next to tail tip C D Femalelateral field at mid-body and areolation (arrows) (Scale bar = 10 microm)
Vol 8(1) 2006 83
E de la Pentildea 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 Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
Vol 8(1) 2006 85
E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
E de la Pentildea 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 Enface view of female lip region D Female lateral field view at mid-body E Excretorysecretory pore F Lateral field and phasmid(arrow) on female tail
84 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
Vol 8(1) 2006 85
E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
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 brzeskiiA P penetrans B P brzeskii C P dunensis sp n (X = DNA ladder 100 bp Promega)
Table 2 Pairwise D2D3 sequence alignment similarity () ofPratylenchus dunensis sp n P brzeskii and P penetrans
Identity
Pratylenchus P brzeskii P penetransdunensis
sp n
P dunensis sp n 100 75 89P brzeskii ndash 100 72P penetrans ndash ndash 100
between outer incisures lateral field partially areolatedthrough whole body rounded to oval spermatheca filledwith round sperm conical tail with 31-39 annules tail tiprounded and smooth with short conspicuous hyaline part(ca 2 microm) phasmid located between inner lateral fieldincisures in posterior half of tail Pratylenchus dunensissp n is further characterised by the abundant presence ofmales (nearly 50 of adults)
As is the case for P dunensis sp n the follow-ing species all have two lip annules and a non-crenatetail P acuticaudatus Braasch amp Dekker 1989 P agilisThorne amp Malek 1968 P alleni Ferris 1961 P angu-latus Siddiqi 1994 P brachyurus (Godfrey 1929) Filip-jev amp Schuurmans Stekhoven 1941 P brzeskii KarssenWaeyenberge amp Moens 2000 P coffeae (Zimmermann
1898) Filipjev amp Schuurmans Stekhoven 1941 P hex-incisus Taylor amp Jenkins 1957 P jordanensis Hashim1984 P loosi Loof 1960 P macrostylus Wu 1971 P ne-glectus (Rensch 1924) Filipjev amp Schuurmans Stekhoven1941 P neobrachyurus Siddiqi 1994 P pseudocoffeaeMizukubo 1992 P scribneri Steiner in Sherbakoff ampStanley 1943 and P silvaticus Brzeski 1998
Morphologically P acuticaudatus P agilis P an-gulatus P brachyurus P hexincisus P jordanensisP macrostylus P neglectus and P scribneri differ fromP dunensis sp n in having an empty spermatheca (malesabsent or very rare) Pratylenchus alleni differs by asmaller stylet (lt15 vs 158-177 microm) and a bluntlyrounded tail with phasmids in the anterior half of thetail (vs conical tail with phasmids posterior to mid-tail)Pratylenchus coffeae and P pseudocoffeae differ in a gen-erally more posterior vulva position (V = 78-83 vs 76-79) a truncate tail (vs conical) elongated spermatheca (vsround to oval) and a well differentiated post uterine branch(vs undifferentiated and short) Pratylenchus loosi differsfrom P dunensis sp n in having a more posterior vulvaposition (V = 79-85 vs 76-79) a broad lateral field withincisures and a relatively shorter ratio tail (c = 18-25 vs13-17) Pratylenchus neobrachyurus is shorter (310-410vs 454-579 microm) and broader (a = 20-31 vs 25-32) with amore posterior vulva (V = 795-83 vs 76-79) fewer tail
Vol 8(1) 2006 85
E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
E de la Pentildea et al
Table 3 Length (bp) of restriction fragments of rDNA Internal Transcribed Spacer regions for Pratylenchus dunensis sp n P brzeskiiand P penetrans
Enzyme Pratylenchus dunensis sp n P penetrans P brzeskii
CfoI 415 280 425 275 610 70EcoRI No restriction 405 295 No restrictionHindIII 400 295 410 290 No restrictionMspI No restriction No restriction 425 255PstI 400 295 395 305 380 300
annules (19-23 vs 31-39) and a non-areolated lateral fieldPratylenchus silvaticus differs from Pratylenchus dunen-sis sp n in head morphology (head not set-off) a compactpharyngeal lobe (vs relatively long) and tail tapering be-fore terminus The ratio between body length and greatestbody diameter is also different between the two species(a = 21-27 vs 25-31) The vulva position in P silvaticusis also slightly more posterior than in P dunensis sp n(V = 80-83 vs 76-79)
Pratylenchus brzeskii shares the same habitat withP dunensis sp n However P brzeskii is longer thanP dunensis sp n (627-737 vs 454-579 microm) has a greaterbody diameter (23-25 vs 15-20 microm) longer pharyngealglands (66-101 vs 33-54 microm) a longer stylet (18-19 vs16-18 microm) and tail length (41-51 vs 30-37 microm) Anotherdifference is found in the lateral field In both species thelateral field at mid-body forms three ridges the middleridge being narrower than the outer ones However inP brzeskii the inner ridge is considerably narrower thanin P dunensis sp n (ridge occupying 40-50 vs 60-80 ofthe distance between the outer lines)
The most common morphotype of P penetrans hasthree lip annuli However some populations of P pene-trans have been reported with two lip annuli (Tarteacute amp Mai1976) Compared with P dunensis sp n P penetransis generally more slender (a = 17-34 vs 254-316) andshows a larger range in the pharyngeal glandbody lengthratio (b = 41-81 vs 38-57) Some tail characteristics arefurther useful characters to distinguish the two speciesthe tail of P penetrans is 21-38 vs 31-39 microm long inP dunensis sp n and there are more tail annules inP dunensis sp n than in P penetrans (31-39 vs 15-27)The lateral field is a further useful character to separatethe species (P penetrans with four equidistant incisuresvs four non-equidistant incisures in P dunensis sp n)
Within the genus Pratylenchus three species are de-scribed as having two lip annules and a crenate tail vizP flakkensis Seinhorst 1968 P estoniensis Ryss 1982and P gibbicaudatus Minawa amp Nozuma 1982 These
species show an obvious crenation of the tail whereasP dunensis sp n has a smooth tail tip Pratylenchusflakkensis also has an elongated spermatheca (vs roundto oval) a smaller number of tail annules (14-26 vs 31-39) and males are uncommon Pratylenchus estoniensishas six lateral lines (vs four) and an empty spermatheca(vs filled with round sperm) and P gibbicaudatus has anempty spermatheca and males absent or rarely present
MOLECULAR CHARACTERISATION
The D2D3 LSU sequences revealed clear differencesbetween P dunensis sp n and P brzeskii and P penetransthe two Pratylenchus species with which the new speciesmight be confused (Table 2) Pratylenchus penetrans isgenetically closer to P dunensis sp n than P brzeskii
The size of the ITS-PCR product was different for thethree species For P dunensis sp n and P penetrans aproduct of nearly 700 bp (695 and 705 bp respectively)was obtained whereas for P brzeskii the PCR yieldeda slightly smaller fragment (680 bp) The ITS-RFLPpatterns obtained after the restriction of the ITS-regionallow the three species to be differentiated (Table 3 Fig 4bottom panel) Pratylenchus brzeskii can be separatedfrom the other two species by CfoI HindIII MspI andPstI Pratylenchus penetrans can be separated from thetwo other species by EcoRI Pratylenchus dunensis sp ncannot be separated with a single digestion but can bedistinguished by comparing the RFLP patterns obtainedwith EcoRI and MspI EcoRI only digested the PCRproduct of P penetrans and MspI only yielded restrictionfragments for P brzeskii
Discussion
The population described in this paper as P dunen-sis sp n was originally identified as P penetrans How-ever further studies of additional molecular data mor-phological characters and morphometrics indicated that
86 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
A new species of Pratylenchus from dune grass
the differences compared to P penetrans justified recog-nition at the species level In addition to the morphol-ogy and morphometrics the sequence comparison of theD2D3 rDNA expansion region clearly separates the newspecies from both P brzeskii and P penetrans We foundat least 25 and 11 nucleotide difference with respect toP brzeskii and P penetrans respectively This rDNA re-gion has been widely used to separate different groups ofnematodes at species level including pratylenchids (Dun-can et al 1999 Handoo et al 2001) The difference be-tween P dunensis sp n and P penetrans is further con-firmed by differences in patterns obtained after endonu-clease restriction of the ITS regions The RFLP profileswe obtained with CfoI HindIII and MspI were the same asthose obtained by Karssen et al (2001) however EcoRIyielded a different pattern for both species (no restrictionvs two bands) and clearly separates the species It is clearthat the number of restriction enzymes used in the firstobservations was not sufficient for species discriminationthe variability of DNA fragments being better estimatedby observing their sequences rather than restriction pat-terns As a consequence our results refute the previouslyreported presence of P penetrans parasitising A arenariain The Netherlands and Seinhorstrsquos (1968) hypothesis thatP penetrans is not native to The Netherlands therefore re-mains valid
The distribution of P dunensis sp n in coastal foredunes with vigorous A arenaria and E farctus sug-gests an adaptation of the species to this habitat and thusmight play a role in the dynamics of the plant commu-nity in coastal dunes Pratylenchus dunensis sp n sharesthe habitat with other migratory or sedentary parasiticnematodes specific for A arenaria such as P brzeskiiMeloidogyne maritima (Jepson 1987) Karssen Van Aelstamp Cook 1998 M duytsi Karssen Van Aelst amp Van derPutten 1998 and Heterodera arenaria Cooper 1955 (seeKarssen et al 1998a b) Recent studies have shown thatPratylenchus spp and H arenaria colonise newly de-posited sand layers thereby indicating that they followroot-growth through the different seasons (Van der Stoelet al 2002) These authors proved the negative influenceof the nematodes on the growth of A arenaria The rela-tionships between the different groups of nematodes andthe host plant are not yet clear However glasshouse tri-als showed that on A arenaria Pratylenchus species arecompeting for food with H arenaria and M maritima(Brinkman 2004) To have a clear picture of the eco-logical processes involving plant-parasitic nematodes in
coastal dunes the effect of P dunensis sp n should there-fore be considered
Acknowledgements
We thank Roel Wagenaar Henk Duyts and Prof Wimvan der Putten at The Netherlands Institute of Ecology(NIOO) Heteren The Netherlands for helping in sam-pling at Groote Keeten This work was funded by EUproject EcoTrain RTN2-2001-0464
References
BRINKMAN P (2004) Interactions among endoparasitic root-feeding nematodes consequences for nematodes and hostplant PhD Thesis Wageningen University Wageningen TheNetherlands 96 pp
BRZESKI MW (1998) Nematodes of Tylenchina in Polandand temperate Europe Warsaw Poland Museum and Insti-tute of Zoology Polish Academy of Sciences 398 pp
COURTNEY WD POLLEY D amp MILLER VI (1955) TAFan improved fixative in nematode technique Plant DiseaseReporter 39 570-571
DE LEY P FELIX MA FRISSE LM NADLER SASTERNBERG PW amp THOMAS WK (1999) Molecularand morphological characterisation of two reproductivelyisolated species with mirror image anatomy (NematodaCephalobidae) Nematology 6 591-612
DE ROOIJ-VAN DER GOES PCEM VAN DER PUTTENWH amp VAN DIJK C (1995) Analysis of nematodesand soil-borne fungi from Ammophila arenaria (marramgrass) in Dutch coastal foredunes by multivariate techniquesEuropean Journal of Plant Pathology 101 149-162
DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRISK amp KAPLAN DT (1999) Molecular and morphologicalanalysis of isolates of Pratylenchus coffeae and closelyrelated species Nematropica 29 61-80
HALL TA (1999) BioEdit a user-friendly biological se-quence alignment editor and analysis program for Windows9598NT Nucleic Acids Symposium Serial 41 95-98
HANDOO ZA CARTA LK amp SKANTAR AM (2001)Morphological and molecular characterisation of Praty-lenchus arlingtoni n sp P convallariae and P fallax (Ne-matoda Pratylenchidae) Nematology 3 607-618
HOOPER DJ amp EVANS K (1993) Extraction identifica-tion and control of plant parasitic nematodes In Evans KTrudgill DL amp Webster JM (Eds) Plant parasitic nema-todes in temperate agriculture Wallingford UK CABI Pub-lishing pp 1-59
KARSSEN G VAN AELST A amp COOK R (1998a) Re-description of the root-knot nematode Meloidogyne maritima
Vol 8(1) 2006 87
E de la Pentildea et al
Jepson 1987 (Nematoda Heteroderidae) a parasite of Am-mophila arenaria (L) Link Nematologica 44 241-253
KARSSEN G VAN AELST A amp VAN DER PUTTEN WH(1998b) Meloidogyne duytsi sp n (Nematoda Heteroderi-dae) a root-knot nematode from Dutch coastal foredunesFundamental and Applied Nematology 21 299-306
KARSSEN G WAEYENBERGE L amp MOENS M (2000)Pratylenchus brzeskii sp nov (Nematoda Pratylenchidae) aroot-lesion nematode from European coastal dunes AnnalesZoologici 50 255-261
KARSSEN G VAN AELST A WAEYENBERGE L ampMOENS M (2001) Observations on Pratylenchus penetransCobb 1917 parasitizing the coastal dune grass Ammophilaarenaria (L) Link in The Netherlands Journal of NematodeMorphology and Systematics 4 1-9
SEINHORST JW (1950) De betekenis van de toestand van degrond voor het optreden van aantasting door het stengelaaltje(Ditylenchus dipsaci Kuumlhn) Tijdschrift voor Plantenziekten10 291-349
SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-515
SELISKAR DM amp HUETTEL RN (1993) Nematodeinvolvement in the dieout of Ammophila breviligulata(Poaceae) on the Mid-Atlantic coastal dunes of the UnitedStates Journal of Coastal Research 9 97-103
TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195
THOMPSON J HIGGINS D amp GIBSON T (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specific gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680
VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
88 Nematology
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VAN DER PUTTEN WH amp PETERS BA (1997) How soil-borne pathogens may affect plant competition Ecology 781785-1795
VAN DER STOEL CD VAN DER PUTTEN WH amp DUYTSH (2002) Development of a negative plant-soil feedback inthe expansion zone of the clonal grass Ammophila arenariafollowing root formation and nematode colonization Journalof Ecology 90 978-988
VERSCHOOR BC PRONK TE DE GOEDE RGM ampBRUSSAARD L (2002) Could plant-feeding nematodes af-fect the competition between grass species during successionin grasslands under restoration management Journal of Ecol-ogy 90 753-761
WAEYENBERGE L RYSS A MOENS M PINOCHETJ amp VRAIN TC (2000) Molecular characterisation of18 Pratylenchus species using rDNA Restriction FragmentLength Polymorphism Nematology 2 135-142
