Nematology 2001 Vol 3(6) 607-618

Morphological and molecular characterisation ofPratylenchus arlingtoni n sp P convallariae and P fallax

(Nematoda Pratylenchidae)Zafar A HANDOO Lynn K CARTA curren and Andrea M SKANTAR

United States Department of Agriculture ARS Nematology Laboratory Beltsville MD 20705 USA

Received 17 October 2000 revised 19 June 2001Accepted for publication23 July 2001

Summary ndash Pratylenchus arlingtoni n sp from the rhizosphere of grasses Poa pratensis and Festuca arundinacea at ArlingtonNational Cemetery VA USA is characterised by six to eight lines in the lateral eld and pyriform to slightly overlapping pharyngealglands Morphological comparisons are made with lesion nematodes having similar morphometrics six lateral lines or crenate tail tipsMolecular sequences of the LS 28S rDNA were generated for the new species as well as P fallax and P convallariae The new speciesdiffers by only 1 from identical sequences found in P fallax and P convallariae

Keywords ndash Festuca arundinacea lesion nematode pathogenicity Poa pratensis Pratylenchus crenatus quarantine

During a search for nematodes in Arlington NationalCemetery Arlington VA USA we discovered a newlesion nematode from soil around grass roots This newspecies described hereunder as Pratylenchus arlingtonin sp had some morphological similarities to crenate-tailed Pratylenchuscrenatus Loof 1960 and Pratylenchusfallax Seinhorst 1968 two other lesion nematode speciesfound in turf in North America and Europe (Yu et al1998)

The purpose of this study was to morphologically andmolecularly describe the new nematode and compare itwith nematodes having similar characters such as P fal-lax and P convallariae Seinhorst 1959 One of thesecharacters was the D3 region of the 28S rDNA whichencodes part of the Large Subunit (LS) rRNA (Baldwinet al 1997) recently used to characterise other speciesof Pratylenchus and their phylogenetic relationships (Al-Banna et al 1997 Duncan et al 1999)

Materials and methods

MORPHOLOGICAL CHARACTERISATION

P arlingtoni n sp was found in turf (Poa pratensisand Festuca arundinacea) soil from Arlington NationalCemetery Arlington VA USA in November 1999

curren Corresponding author e-mail cartalbaarsusdagov

P convallariae and P fallax were isolated from inter-cepted lily of the valley shipments (March 1999 and Jan-uary 2000 respectively from a company in Europe) af-ter collection at JFK International Airport Jamaica NewYork by Bernice Medina and dispatched to us for identi -cation by Alan Towson USDA Animal and Plant HealthInspection Service (APHIS)

Nematodes were extracted from soil with sieves or fromchopped infested roots over lter paper in Baermannfunnels Specimens were then handpicked and xed in3 formalin Some specimens were studied in xativeon temporary slide mounts others in permanent glycer-ine mounts (Golden 1990) or viewed live with or with-out 5 mM sodium azide for narcotisation on an agar pad(Stiernagle 1999) Examinations were made with a com-pound light microscope and morphometric data obtainedwith an ocular micrometer All measurements are in mi-crometers (sup1m) unless otherwise speci ed Light micro-scopic images of live and xed nematodes were takenwith the Bioquant ver 32 imaging system (BiometricsInc Nashville TN USA) on a Leitz Ortholux micro-scope Differential interference contrast (DIC) images oflive nematodes were taken with an imaging system em-ploying Image-Pro Plus ver 30 (I-Cube Image Analy-sisImage Processing Crofton MD USA) on a Zeiss Ul-traphot II microscope equipped with DIC optics Nema-

cdeg Koninklijke Brill NV Leiden 2001 607

ZA Handoo et al

todes were processed for scanning electron microscopy(SEM) in room temperature 005 phosphate-buffered (pH68) 3 glutaraldehyde (12 h) and 2 osmium tetroxide(2 h) and viewed after ethanol dehydration critical-pointdrying and gold-palladium coating on a Jeol JSM-T300microscope at 20 kV

TEMPLATE PREPARATION

Nematode extracts were prepared by the procedureof Williams et al (1992) A single nematode was pla-ced in 10 sup1l of digestion buffer (10 mM Tris pH 8225 mM MgCl2I 50 mM KCl 045 Tween 20 005gelatin 60 sup1gml proteinase K) and frozen at iexcl70plusmnC for15 min to several days The extracts were thawed over-laid with a drop of mineral oil and warmed to 60plusmnC for1 h Proteinase K was denatured by heating to 95plusmnC for15 min

AMPLIFICATION AND SEQUENCING

The D3 expansion region of 28S rDNA (345 nucleotidebase pairs (bp) raw sequence) was ampli ed separatelyfrom two adult nematodes of each species using hot-start reactions as described by Chou et al (1992) with thefollowing modi cations Manufacturer-supplied Display-TAQ buffer (PGC Scienti c Gaithersburg MD USA)250 sup1M dNTPs 4 mM MgCl2 and 600 sup1M of each ri-bosomal DNA primer originally designed by W KelleyThomas University of Missouri Kansas City MO USAPrimers D3A (50-GACCCGTCTTGAAACACGGA-3 0)and D3B (50-TCGGAAGGAACCAGCTACTA-30) (Bald-win et al 1997) were added to the bottom of 05 ml thin-wall microcentrifuge tubes A drop (ca 25 sup1l) of paraf- n wax was overlaid and allowed to cool forming aneven barrier The remaining TAQ buffer template and Dis-play TAQ were then layered on top of the wax Cyclingconditions were 94plusmnC 3 min (to allow hot start) 94plusmnC1 min 52plusmnC 1 min 72plusmnC 1 min pound35 cycles 72plusmnC10 min Reactions were analysed by gel electrophore-sis

DNA sequences were obtained by sequencing PCRproducts directly or by sequencing cloned PCR productsFor direct sequencing whole nematode extracts (10 sup1l)were included in the PCR reactions to generate a suf cientamount of PCR product Prior to sequencing the DNAwas puri ed using the Qiaquick PCR puri cation kit (Qi-agen ValenciaCA USA) Sequencingreactions included100 ng PCR product and 32 pmol D3A or D3B primer Togenerate cloned PCR products for sequencing2 sup1l nema-

tode extract was included in each PCR reaction The re-sultant PCR products were cloned into the vector pCR21using the Topo-TA Cloning kit (InvitrogenCarlsbad CAUSA) Plasmid DNA was puri ed from bacterial culturesusing Wizard Preps (Promega Madison WI USA) Thesequencing reactions contained 200 ng plasmid templateand the M13 forward or M13 reverse primers All BigDyeTerminator cycle sequencingwas performed using an ABI377 Sequencer (PE-Applied Biosystems Foster City CAUSA)

Negative controls included reactions with water or amock extract (no nematode) instead of DNA A reac-tion containing5 ng Meloidogynejavanica genomic DNAwas included as a positive control To con rm the au-thenticity of the sequences obtained PCR ampli cationand DNA sequencing were performed on two individu-als from the same nematode population To account forthe possibility of PCR-generated errors in the cloned PCRproducts we compared the sequences from two or moreclones obtained from the same nematode extract If ambi-guities were detected between clones sequences deriveddirectly from PCR products were used to resolve the con- ict

The sequences for P arlingtoni n sp P convallariaeand P fallax have been deposited in the GenBank data-base (National Center for BiotechnologyInformationNa-tional Library of Medicine National Institute of HealthBethesda MD USA httpwwwncbinlmnihgov) asAF307328 AF196351 and AF264181 respectively

ALIGNMENT

From the 345 bp of raw sequence 305 bp were usedin the nal alignment The new sequences were alignedwith the Clustal W (ver 14) program (Clustal W WWWService at the European Bioinformatics Institute Ro-drigo Lopez Services Programme httpwww2ebiacukclustalw Thompson et al 1994) with all Pratylenchussequences in the GenBank database The closest sequenceto the new species from the GenBank database is alsoshown in the alignment provided here (Fig 5) Positionsare numbered where 1 corresponds to number 3324 ofthe Caenorhabditis elegans 28S rRNA gene (Ellis et al1986 Al-Banna et al 1997) The number and posi-tion of nucleotide differences among the four taxa werenoted

608 Nematology

ZA Handoo et al

todes were processed for scanning electron microscopy(SEM) in room temperature 005 phosphate-buffered (pH68) 3 glutaraldehyde (12 h) and 2 osmium tetroxide(2 h) and viewed after ethanol dehydration critical-pointdrying and gold-palladium coating on a Jeol JSM-T300microscope at 20 kV

TEMPLATE PREPARATION

Nematode extracts were prepared by the procedureof Williams et al (1992) A single nematode was pla-ced in 10 sup1l of digestion buffer (10 mM Tris pH 8225 mM MgCl2I 50 mM KCl 045 Tween 20 005gelatin 60 sup1gml proteinase K) and frozen at iexcl70plusmnC for15 min to several days The extracts were thawed over-laid with a drop of mineral oil and warmed to 60plusmnC for1 h Proteinase K was denatured by heating to 95plusmnC for15 min

AMPLIFICATION AND SEQUENCING

The D3 expansion region of 28S rDNA (345 nucleotidebase pairs (bp) raw sequence) was ampli ed separatelyfrom two adult nematodes of each species using hot-start reactions as described by Chou et al (1992) with thefollowing modi cations Manufacturer-supplied Display-TAQ buffer (PGC Scienti c Gaithersburg MD USA)250 sup1M dNTPs 4 mM MgCl2 and 600 sup1M of each ri-bosomal DNA primer originally designed by W KelleyThomas University of Missouri Kansas City MO USAPrimers D3A (50-GACCCGTCTTGAAACACGGA-3 0)and D3B (50-TCGGAAGGAACCAGCTACTA-30) (Bald-win et al 1997) were added to the bottom of 05 ml thin-wall microcentrifuge tubes A drop (ca 25 sup1l) of paraf- n wax was overlaid and allowed to cool forming aneven barrier The remaining TAQ buffer template and Dis-play TAQ were then layered on top of the wax Cyclingconditions were 94plusmnC 3 min (to allow hot start) 94plusmnC1 min 52plusmnC 1 min 72plusmnC 1 min pound35 cycles 72plusmnC10 min Reactions were analysed by gel electrophore-sis

DNA sequences were obtained by sequencing PCRproducts directly or by sequencing cloned PCR productsFor direct sequencing whole nematode extracts (10 sup1l)were included in the PCR reactions to generate a suf cientamount of PCR product Prior to sequencing the DNAwas puri ed using the Qiaquick PCR puri cation kit (Qi-agen ValenciaCA USA) Sequencingreactions included100 ng PCR product and 32 pmol D3A or D3B primer Togenerate cloned PCR products for sequencing2 sup1l nema-

tode extract was included in each PCR reaction The re-sultant PCR products were cloned into the vector pCR21using the Topo-TA Cloning kit (InvitrogenCarlsbad CAUSA) Plasmid DNA was puri ed from bacterial culturesusing Wizard Preps (Promega Madison WI USA) Thesequencing reactions contained 200 ng plasmid templateand the M13 forward or M13 reverse primers All BigDyeTerminator cycle sequencingwas performed using an ABI377 Sequencer (PE-Applied Biosystems Foster City CAUSA)

Negative controls included reactions with water or amock extract (no nematode) instead of DNA A reac-tion containing5 ng Meloidogynejavanica genomic DNAwas included as a positive control To con rm the au-thenticity of the sequences obtained PCR ampli cationand DNA sequencing were performed on two individu-als from the same nematode population To account forthe possibility of PCR-generated errors in the cloned PCRproducts we compared the sequences from two or moreclones obtained from the same nematode extract If ambi-guities were detected between clones sequences deriveddirectly from PCR products were used to resolve the con- ict

The sequences for P arlingtoni n sp P convallariaeand P fallax have been deposited in the GenBank data-base (National Center for BiotechnologyInformationNa-tional Library of Medicine National Institute of HealthBethesda MD USA httpwwwncbinlmnihgov) asAF307328 AF196351 and AF264181 respectively

ALIGNMENT

From the 345 bp of raw sequence 305 bp were usedin the nal alignment The new sequences were alignedwith the Clustal W (ver 14) program (Clustal W WWWService at the European Bioinformatics Institute Ro-drigo Lopez Services Programme httpwww2ebiacukclustalw Thompson et al 1994) with all Pratylenchussequences in the GenBank database The closest sequenceto the new species from the GenBank database is alsoshown in the alignment provided here (Fig 5) Positionsare numbered where 1 corresponds to number 3324 ofthe Caenorhabditis elegans 28S rRNA gene (Ellis et al1986 Al-Banna et al 1997) The number and posi-tion of nucleotide differences among the four taxa werenoted

608 Nematology

Pratylenchus arlingtoni n sp

Table 1 Morphometrics of Pratylenchus arlingtoni n sp females (All measurements in sup1m) Values are means sect standard deviationValues in parentheses indicate ranges

n D 20 Holotype Paratypes

L 423 455 sect 37 (405 - 535)a 248 27 sect 3 (21 - 33)b 40 44 sect 04 (41 - 53)c 206 22 sect 2 (18 - 28)V 815 823 sect 12 (81 - 86)Stylet length 17 168 sect 05 (16 - 175)c0 20 13 sect 01 (11 - 15)Tail annule number 21 209 sect 18 (19 - 25)Lateral line number ndash ndash (6 - 8) aerolate extremesPost-uterine sac 25 194 sect 28 (15 - 25)Vulval width 15 159 sect 11 (14 - 175)Vulva-Anus distance 53 559 sect 71 (43 - 70)Post-uterine sacVulval width 17 ndash (15 - 25)Post-uterine sacVulva-anus pound 100 47 ndash (28 - 42)Pharyngeal overlap ndash 20 sect 78 (6 - 31) pyriform to overlapMalesspermatheca ndash Not foundovalLip number shape ndash 3 slightly offsetExcretory pore position ndash Pharyngeal-intestinaljunctionExcretory pore-lips ndash 70 sect 10 (48 - 85)Body diam 15 17 sect 14 (15 - 20)Pharynx length ndash 101 sect 35 (93 - 107)Tail length ndash 21 sect 18 (15 - 23)Stylet knob height ndash 27 sect 03 (25 - 30)Stylet knob width ndash 46 sect 02 (45 - 50)Stylet knob widthheight ndash 17 sect 01 (16 - 18)Head diam ndash 81 C 04 (75 - 90)Head height ndash 28 sect 03 (25 - 30)Head diamheight ndash 28 sect 03 (26 - 32)

Pratylenchus arlingtonicurren n sp(Figs 1 - 3 5)

MEASUREMENTS

Females

See Table 1

DESCRIPTION

Female

Body vermiform with some tapering at extremities Lipregion slightly offset three annules Head framework ex-tending inward for two or three annulesAnterior body an-nules measuring 12 sup1m tail annules measuring 09 sup1mStylet knobs broadly rounded with anterior outer edges

From Arlington VA USA type locality

directed slightly forward Dorsal pharyngeal gland open-ing at 2-3 sup1m behind the stylet knobs Lateral eld begin-ning behind the level of the stylet as four narrow crenatelines widening to ve by the level of the median bulband six to eight by the anterior intestine level through tothe vulva Four lines extending to the phasmid just pos-terior to the vulva after which three lines extend a fewannules short of the tail tip Lateral eld sometimes aero-lated at the extremes Pharyngeal glands pyriform in ap-proximately 25 of the specimens or slightly overlappingin others Excretory pore and canal located within an areaslightly above or below the pharyngeal-intestinal junctionVulva elevated the slit extending inward about 70 of thebody diam Anterior gonad with a single row of oocytesand extending forward for nearly three times the distancebetween the vulva and anus An egg (36-cell) within thebody measuring 75pound21 sup1m another (4-cell) 64pound14 sup1m

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ZA Handoo et al

Oval spermatheca 17 sup1m in length with apparent spermcells of 14 - 22 sup1m diam 74 sup1m anterior to the vulva(one specimen out of at least 200) Distance from vulvato spermatheca 120 of the distance between vulva andanus Post-uterine sac generally undifferentiated with adiscernible tip cell or four columnar cells at the dorsalarch sometimes present Phasmid located 10 to 15 annulesabove the tail tip Tail terminus coarsely annulated withvariable shape from conoid clavate to truncate or some-times bi d

Male

Not found

TYPE HOST AND LOCALITY

Roots of turf (Poa pratensis blue grass and Festucaarundinacea tall fescue) under Quercus spp (oaks) atOld Section 27 ArlingtonCemetery ArlingtonVA USAThis section of the cemetery was from the old ArlingtonFarm uncultivated since the 1800s

TYPE SPECIMENS

Holotype

Slide T-543p deposited in the United States Depart-ment of Agriculture Nematode Collection (USDANC)Beltsville MD USA

Paratypes (females)

Same data and repository as holotype Slides T-4918p-4921p with 15 mixed stage females and slides T-4922p-4924p with single adult females Others deposited inthe nematode collections at The University of Califor-nia Davis Davis CA USA The Canadian Food Inspec-tion Agency Centre for Plant Quarantine Pests OntarioCanada Rothamsted Experimental Station RothamstedUK Museacuteum National drsquoHistoire Naturelle Laboratoiredes Vers Paris France Wageningen University and Re-search Centre Landbouwhogeschool Wageningen TheNetherlands and the Zoological Institute Russian Acad-emy of Sciences St Petersburg Russia

DIAGNOSIS AND RELATIONSHIPS

P arlingtoni n sp is unique among lesion nematodespecies in having six to eight lateral lines on specimensfrom the pharyngeal to the vulval region and pharyngealglands with a pyriform basal bulb to a shallow overlap It

Fig 1 Pratylenchus arlingtoni n sp (lateral views) A Body BLateral eld midbody C Head and pharynx D - F Tail

also has a crenate tail tip an elevated vulva no observedmales and a very rare oval spermatheca

P arlingtoni n sp shares a variably crenate tail withP cerealis Haque 1965 P convallariae Seinhorst 1959

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Pratylenchus arlingtoni n sp

Fig 2 Pratylenchus arlingtoni n sp Scanning electron micrographs A B Lip region (face view) with and without amphid secretionC Lip region (lateral view) D Body E Tail and phasmid opening (arrow) F Lateral eld G Tail region (lateral view)

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ZA Handoo et al

Fig 3 Pratylenchus arlingtoni n sp A Head end (lateral) with median bulb light microscopy (LM) B Pharynx pyriform basal bulb(ventrolateralview) LM C Female tail (lateral view) LM D Head (lateral view) Differential interferencecontrast (DIC) E Lateral eld midbody LM F Pharyngeal-intestinal junction (pij) and excretory canal (exc) (lateral view) DIC G Lateral eld mid-bodyDIC H Spermatheca with sperm (lateral view) DIC I Female gonad with vulva egg post-uterine sac (lateral view) DIC

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Pratylenchus arlingtoni n sp

P crenatus Loof 1960 P fallax Seinhorst 1968 P gibbi-caudatus Minagawa 1982 P gutierrezi Golden Loacutepez ampViacutelchez 1992 P pratensis (de Man 1880) Filipjev 1936P pseudofallaxCafeacute-Filho amp Huang 1989 P roseus Za-rina amp Maqbool 1998 P teres Khan amp Singh 1974 andP yassini Zeidan amp Geraert 1991

P crenatus P roseus and P teres may also have upto six lateral lines P arlingtoni n sp is morphologicallymost similar to P crenatus but differs by longer pharynx(93 - 107 vs 57 - 78 sup1m) pyriform to slightly overlappingbasal pharyngealglands (6 - 31 vs 17 - 38 sup1m) somewhatlower b value (41 - 53 vs 49 - 70 sup1m in P crenatusLoof 1960 Torres amp Chaves 1999 Urek 1999) smallerpost-uterine sac length relative to vulval-anal distance(PUSVA pound 100 28 - 42 vs 40 - 50) lower c0 value(11 - 15 vs 16 - 29) more elevated vulva and sixto eight lateral lines compared to four to six lines Theexcretory pore in P arlingtoni n sp is often below thepharyngeal-intestinal junction (p-ij) and is above thep-ij in P crenatus P arlingtoni n sp lacks a vulvalmembrane has a longer stylet (16 - 175 vs 15 - 16 sup1m)and a shorter pharyngealoverlap (6 - 31 vs 118 - 130 sup1m)compared to P roseus P arlingtoni n sp differs fromP teres by more posterior vulva (V = 81 - 86 vs 69 - 78)fewer tail annules (19 - 25 vs 24 - 30) shorter pharyngealoverlap (6 - 31 vs 58 sup1m) longer PUSVA pound 100 (28 - 42vs 18) and six to eight lateral lines rather than only six

When P arlingtoni n sp is compared to both P conva-llariae and P fallax it has a lower b value (41 - 53 vs 60- 90 and 52 - 67 respectively) and more posterior vulva(V = 81 - 86 vs 78 - 81 and 77 - 81 respectively) lacksmales and has two to four more lateral lines P arlingtonin sp also has more tail annules (19 - 25 vs 16 - 19) thanP convallariae The new species has a longer stylet (160 -175 vs 150 - 155 sup1m) more offset lip region and coarsertail annulation than P fallax Differences from P fallaxalso apply to P pseudofallax(Cafeacute-Filho amp Huang 1989)P arlingtoni n sp differs from P penetrans by having acrenate tail in all specimens lack of observedmales lowerb value (41 - 53 vs 53 - 79) shorter pharyngeal overlap(6 - 31 vs 32 - 65 sup1m) and longer PUSVW (15 - 25 vs10 - 15)

Observations on intercepted Pratylenchus fallaxand P convallariae

P fallax is common in sandy or sandy-peat soils (Sein-horst 1977) around grass and ornamentals of many Eu-ropean countries (Webb 1990) as well as the Canadian

provinces of Quebec and Ontario (Yu et al 1998) How-ever P fallax was found in the United States only in 1974on strawberries in Iowa (Norton 1984) Based on its geo-graphic distributionand pathogenicityto barley and maizein Europe (Corbett 1970 1972) it is underUS quarantinerestriction (Joseph Cavey pers comm)

A morphologicallyrelated species P convallariaeSein-horst 1959 is a non-endemic quality pest common inlight sandy soils (Seinhorst 1959) from Convallaria ma-jalis (lily of the valley) shipments from northern Euro-pean countries to the United States It was detected at leastsix times at the USDA Nematology Laboratory from late1998 to late 2000Because of its limited geographicdistri-bution but long-termpresence in the United States it is notsubject to regulatory action (Joseph Cavey pers comm)

P fallax was originally distinguished from P convalla-riae by its shorter body length narrower more numeroustail annules and lower male to female ratio (Seinhorst1968) P fallax was synonymisedby Frederick and Tarjan(1989) with P cerealis Haque 1965 because of the beliefshared by Loof (1978) that the attened condition of thetype specimens accounted for an arti cially much lowera ratio However the original description of P fallax alsodistinguished it from P cerealis by a longer body length(042 - 056 vs 039 - 043 mm) more posterior excretorypore (between nerve ring and pharyngeal-intestinal valvevs at level of median bulb in P cerealis) a longer tail andmore obscure atter lip annules (Seinhorst 1968)

Recently both P fallax and P convallariae were de-tected in a shipment of C majalis from France throughthe Netherlands and destined for the state of Ohio USAMountedspecimens of original paratypes of P fallaxwerecompared with P convallariae from a previous intercep-tion Intermediate morphologicalforms were also noticedbetween the typical P convallariae and P fallax Pho-tographs and supplemental measurements of representa-tive members of each species are provided and comparedwith previous descriptions

The P fallax population from France had typical spe-cies-diagnostic characters of crenate tail terminus in allmembers of the population (compared to P penetrans)oblique central lateral eld striations body length (048- 505 vs 050 - 056 mm in original description vs 058 -061 mm for P convallariae) rounded to anchor-shapedstylet knobs compared to more tulip-shaped knobs inP convallariae tail annule numbers (19 - 26 vs 16 - 19in P convallariae) and rare males to distinguish it fromP convallariae (Table 2 Fig 4) or P penetrans wheremales are more numerous Stylet lengths in this popula-

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ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

ZA Handoo et al

Oval spermatheca 17 sup1m in length with apparent spermcells of 14 - 22 sup1m diam 74 sup1m anterior to the vulva(one specimen out of at least 200) Distance from vulvato spermatheca 120 of the distance between vulva andanus Post-uterine sac generally undifferentiated with adiscernible tip cell or four columnar cells at the dorsalarch sometimes present Phasmid located 10 to 15 annulesabove the tail tip Tail terminus coarsely annulated withvariable shape from conoid clavate to truncate or some-times bi d

Male

Not found

TYPE HOST AND LOCALITY

Roots of turf (Poa pratensis blue grass and Festucaarundinacea tall fescue) under Quercus spp (oaks) atOld Section 27 ArlingtonCemetery ArlingtonVA USAThis section of the cemetery was from the old ArlingtonFarm uncultivated since the 1800s

TYPE SPECIMENS

Holotype

Slide T-543p deposited in the United States Depart-ment of Agriculture Nematode Collection (USDANC)Beltsville MD USA

Paratypes (females)

Same data and repository as holotype Slides T-4918p-4921p with 15 mixed stage females and slides T-4922p-4924p with single adult females Others deposited inthe nematode collections at The University of Califor-nia Davis Davis CA USA The Canadian Food Inspec-tion Agency Centre for Plant Quarantine Pests OntarioCanada Rothamsted Experimental Station RothamstedUK Museacuteum National drsquoHistoire Naturelle Laboratoiredes Vers Paris France Wageningen University and Re-search Centre Landbouwhogeschool Wageningen TheNetherlands and the Zoological Institute Russian Acad-emy of Sciences St Petersburg Russia

DIAGNOSIS AND RELATIONSHIPS

P arlingtoni n sp is unique among lesion nematodespecies in having six to eight lateral lines on specimensfrom the pharyngeal to the vulval region and pharyngealglands with a pyriform basal bulb to a shallow overlap It

Fig 1 Pratylenchus arlingtoni n sp (lateral views) A Body BLateral eld midbody C Head and pharynx D - F Tail

also has a crenate tail tip an elevated vulva no observedmales and a very rare oval spermatheca

P arlingtoni n sp shares a variably crenate tail withP cerealis Haque 1965 P convallariae Seinhorst 1959

610 Nematology

Pratylenchus arlingtoni n sp

Fig 2 Pratylenchus arlingtoni n sp Scanning electron micrographs A B Lip region (face view) with and without amphid secretionC Lip region (lateral view) D Body E Tail and phasmid opening (arrow) F Lateral eld G Tail region (lateral view)

Vol 3(6) 2001 611

ZA Handoo et al

Fig 3 Pratylenchus arlingtoni n sp A Head end (lateral) with median bulb light microscopy (LM) B Pharynx pyriform basal bulb(ventrolateralview) LM C Female tail (lateral view) LM D Head (lateral view) Differential interferencecontrast (DIC) E Lateral eld midbody LM F Pharyngeal-intestinal junction (pij) and excretory canal (exc) (lateral view) DIC G Lateral eld mid-bodyDIC H Spermatheca with sperm (lateral view) DIC I Female gonad with vulva egg post-uterine sac (lateral view) DIC

612 Nematology

Pratylenchus arlingtoni n sp

P crenatus Loof 1960 P fallax Seinhorst 1968 P gibbi-caudatus Minagawa 1982 P gutierrezi Golden Loacutepez ampViacutelchez 1992 P pratensis (de Man 1880) Filipjev 1936P pseudofallaxCafeacute-Filho amp Huang 1989 P roseus Za-rina amp Maqbool 1998 P teres Khan amp Singh 1974 andP yassini Zeidan amp Geraert 1991

P crenatus P roseus and P teres may also have upto six lateral lines P arlingtoni n sp is morphologicallymost similar to P crenatus but differs by longer pharynx(93 - 107 vs 57 - 78 sup1m) pyriform to slightly overlappingbasal pharyngealglands (6 - 31 vs 17 - 38 sup1m) somewhatlower b value (41 - 53 vs 49 - 70 sup1m in P crenatusLoof 1960 Torres amp Chaves 1999 Urek 1999) smallerpost-uterine sac length relative to vulval-anal distance(PUSVA pound 100 28 - 42 vs 40 - 50) lower c0 value(11 - 15 vs 16 - 29) more elevated vulva and sixto eight lateral lines compared to four to six lines Theexcretory pore in P arlingtoni n sp is often below thepharyngeal-intestinal junction (p-ij) and is above thep-ij in P crenatus P arlingtoni n sp lacks a vulvalmembrane has a longer stylet (16 - 175 vs 15 - 16 sup1m)and a shorter pharyngealoverlap (6 - 31 vs 118 - 130 sup1m)compared to P roseus P arlingtoni n sp differs fromP teres by more posterior vulva (V = 81 - 86 vs 69 - 78)fewer tail annules (19 - 25 vs 24 - 30) shorter pharyngealoverlap (6 - 31 vs 58 sup1m) longer PUSVA pound 100 (28 - 42vs 18) and six to eight lateral lines rather than only six

When P arlingtoni n sp is compared to both P conva-llariae and P fallax it has a lower b value (41 - 53 vs 60- 90 and 52 - 67 respectively) and more posterior vulva(V = 81 - 86 vs 78 - 81 and 77 - 81 respectively) lacksmales and has two to four more lateral lines P arlingtonin sp also has more tail annules (19 - 25 vs 16 - 19) thanP convallariae The new species has a longer stylet (160 -175 vs 150 - 155 sup1m) more offset lip region and coarsertail annulation than P fallax Differences from P fallaxalso apply to P pseudofallax(Cafeacute-Filho amp Huang 1989)P arlingtoni n sp differs from P penetrans by having acrenate tail in all specimens lack of observedmales lowerb value (41 - 53 vs 53 - 79) shorter pharyngeal overlap(6 - 31 vs 32 - 65 sup1m) and longer PUSVW (15 - 25 vs10 - 15)

Observations on intercepted Pratylenchus fallaxand P convallariae

P fallax is common in sandy or sandy-peat soils (Sein-horst 1977) around grass and ornamentals of many Eu-ropean countries (Webb 1990) as well as the Canadian

provinces of Quebec and Ontario (Yu et al 1998) How-ever P fallax was found in the United States only in 1974on strawberries in Iowa (Norton 1984) Based on its geo-graphic distributionand pathogenicityto barley and maizein Europe (Corbett 1970 1972) it is underUS quarantinerestriction (Joseph Cavey pers comm)

A morphologicallyrelated species P convallariaeSein-horst 1959 is a non-endemic quality pest common inlight sandy soils (Seinhorst 1959) from Convallaria ma-jalis (lily of the valley) shipments from northern Euro-pean countries to the United States It was detected at leastsix times at the USDA Nematology Laboratory from late1998 to late 2000Because of its limited geographicdistri-bution but long-termpresence in the United States it is notsubject to regulatory action (Joseph Cavey pers comm)

P fallax was originally distinguished from P convalla-riae by its shorter body length narrower more numeroustail annules and lower male to female ratio (Seinhorst1968) P fallax was synonymisedby Frederick and Tarjan(1989) with P cerealis Haque 1965 because of the beliefshared by Loof (1978) that the attened condition of thetype specimens accounted for an arti cially much lowera ratio However the original description of P fallax alsodistinguished it from P cerealis by a longer body length(042 - 056 vs 039 - 043 mm) more posterior excretorypore (between nerve ring and pharyngeal-intestinal valvevs at level of median bulb in P cerealis) a longer tail andmore obscure atter lip annules (Seinhorst 1968)

Recently both P fallax and P convallariae were de-tected in a shipment of C majalis from France throughthe Netherlands and destined for the state of Ohio USAMountedspecimens of original paratypes of P fallaxwerecompared with P convallariae from a previous intercep-tion Intermediate morphologicalforms were also noticedbetween the typical P convallariae and P fallax Pho-tographs and supplemental measurements of representa-tive members of each species are provided and comparedwith previous descriptions

The P fallax population from France had typical spe-cies-diagnostic characters of crenate tail terminus in allmembers of the population (compared to P penetrans)oblique central lateral eld striations body length (048- 505 vs 050 - 056 mm in original description vs 058 -061 mm for P convallariae) rounded to anchor-shapedstylet knobs compared to more tulip-shaped knobs inP convallariae tail annule numbers (19 - 26 vs 16 - 19in P convallariae) and rare males to distinguish it fromP convallariae (Table 2 Fig 4) or P penetrans wheremales are more numerous Stylet lengths in this popula-

Vol 3(6) 2001 613

ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

Pratylenchus arlingtoni n sp

Fig 2 Pratylenchus arlingtoni n sp Scanning electron micrographs A B Lip region (face view) with and without amphid secretionC Lip region (lateral view) D Body E Tail and phasmid opening (arrow) F Lateral eld G Tail region (lateral view)

Vol 3(6) 2001 611

ZA Handoo et al

Fig 3 Pratylenchus arlingtoni n sp A Head end (lateral) with median bulb light microscopy (LM) B Pharynx pyriform basal bulb(ventrolateralview) LM C Female tail (lateral view) LM D Head (lateral view) Differential interferencecontrast (DIC) E Lateral eld midbody LM F Pharyngeal-intestinal junction (pij) and excretory canal (exc) (lateral view) DIC G Lateral eld mid-bodyDIC H Spermatheca with sperm (lateral view) DIC I Female gonad with vulva egg post-uterine sac (lateral view) DIC

612 Nematology

Pratylenchus arlingtoni n sp

P crenatus Loof 1960 P fallax Seinhorst 1968 P gibbi-caudatus Minagawa 1982 P gutierrezi Golden Loacutepez ampViacutelchez 1992 P pratensis (de Man 1880) Filipjev 1936P pseudofallaxCafeacute-Filho amp Huang 1989 P roseus Za-rina amp Maqbool 1998 P teres Khan amp Singh 1974 andP yassini Zeidan amp Geraert 1991

P crenatus P roseus and P teres may also have upto six lateral lines P arlingtoni n sp is morphologicallymost similar to P crenatus but differs by longer pharynx(93 - 107 vs 57 - 78 sup1m) pyriform to slightly overlappingbasal pharyngealglands (6 - 31 vs 17 - 38 sup1m) somewhatlower b value (41 - 53 vs 49 - 70 sup1m in P crenatusLoof 1960 Torres amp Chaves 1999 Urek 1999) smallerpost-uterine sac length relative to vulval-anal distance(PUSVA pound 100 28 - 42 vs 40 - 50) lower c0 value(11 - 15 vs 16 - 29) more elevated vulva and sixto eight lateral lines compared to four to six lines Theexcretory pore in P arlingtoni n sp is often below thepharyngeal-intestinal junction (p-ij) and is above thep-ij in P crenatus P arlingtoni n sp lacks a vulvalmembrane has a longer stylet (16 - 175 vs 15 - 16 sup1m)and a shorter pharyngealoverlap (6 - 31 vs 118 - 130 sup1m)compared to P roseus P arlingtoni n sp differs fromP teres by more posterior vulva (V = 81 - 86 vs 69 - 78)fewer tail annules (19 - 25 vs 24 - 30) shorter pharyngealoverlap (6 - 31 vs 58 sup1m) longer PUSVA pound 100 (28 - 42vs 18) and six to eight lateral lines rather than only six

When P arlingtoni n sp is compared to both P conva-llariae and P fallax it has a lower b value (41 - 53 vs 60- 90 and 52 - 67 respectively) and more posterior vulva(V = 81 - 86 vs 78 - 81 and 77 - 81 respectively) lacksmales and has two to four more lateral lines P arlingtonin sp also has more tail annules (19 - 25 vs 16 - 19) thanP convallariae The new species has a longer stylet (160 -175 vs 150 - 155 sup1m) more offset lip region and coarsertail annulation than P fallax Differences from P fallaxalso apply to P pseudofallax(Cafeacute-Filho amp Huang 1989)P arlingtoni n sp differs from P penetrans by having acrenate tail in all specimens lack of observedmales lowerb value (41 - 53 vs 53 - 79) shorter pharyngeal overlap(6 - 31 vs 32 - 65 sup1m) and longer PUSVW (15 - 25 vs10 - 15)

Observations on intercepted Pratylenchus fallaxand P convallariae

P fallax is common in sandy or sandy-peat soils (Sein-horst 1977) around grass and ornamentals of many Eu-ropean countries (Webb 1990) as well as the Canadian

provinces of Quebec and Ontario (Yu et al 1998) How-ever P fallax was found in the United States only in 1974on strawberries in Iowa (Norton 1984) Based on its geo-graphic distributionand pathogenicityto barley and maizein Europe (Corbett 1970 1972) it is underUS quarantinerestriction (Joseph Cavey pers comm)

A morphologicallyrelated species P convallariaeSein-horst 1959 is a non-endemic quality pest common inlight sandy soils (Seinhorst 1959) from Convallaria ma-jalis (lily of the valley) shipments from northern Euro-pean countries to the United States It was detected at leastsix times at the USDA Nematology Laboratory from late1998 to late 2000Because of its limited geographicdistri-bution but long-termpresence in the United States it is notsubject to regulatory action (Joseph Cavey pers comm)

P fallax was originally distinguished from P convalla-riae by its shorter body length narrower more numeroustail annules and lower male to female ratio (Seinhorst1968) P fallax was synonymisedby Frederick and Tarjan(1989) with P cerealis Haque 1965 because of the beliefshared by Loof (1978) that the attened condition of thetype specimens accounted for an arti cially much lowera ratio However the original description of P fallax alsodistinguished it from P cerealis by a longer body length(042 - 056 vs 039 - 043 mm) more posterior excretorypore (between nerve ring and pharyngeal-intestinal valvevs at level of median bulb in P cerealis) a longer tail andmore obscure atter lip annules (Seinhorst 1968)

Recently both P fallax and P convallariae were de-tected in a shipment of C majalis from France throughthe Netherlands and destined for the state of Ohio USAMountedspecimens of original paratypes of P fallaxwerecompared with P convallariae from a previous intercep-tion Intermediate morphologicalforms were also noticedbetween the typical P convallariae and P fallax Pho-tographs and supplemental measurements of representa-tive members of each species are provided and comparedwith previous descriptions

The P fallax population from France had typical spe-cies-diagnostic characters of crenate tail terminus in allmembers of the population (compared to P penetrans)oblique central lateral eld striations body length (048- 505 vs 050 - 056 mm in original description vs 058 -061 mm for P convallariae) rounded to anchor-shapedstylet knobs compared to more tulip-shaped knobs inP convallariae tail annule numbers (19 - 26 vs 16 - 19in P convallariae) and rare males to distinguish it fromP convallariae (Table 2 Fig 4) or P penetrans wheremales are more numerous Stylet lengths in this popula-

Vol 3(6) 2001 613

ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

ZA Handoo et al

Fig 3 Pratylenchus arlingtoni n sp A Head end (lateral) with median bulb light microscopy (LM) B Pharynx pyriform basal bulb(ventrolateralview) LM C Female tail (lateral view) LM D Head (lateral view) Differential interferencecontrast (DIC) E Lateral eld midbody LM F Pharyngeal-intestinal junction (pij) and excretory canal (exc) (lateral view) DIC G Lateral eld mid-bodyDIC H Spermatheca with sperm (lateral view) DIC I Female gonad with vulva egg post-uterine sac (lateral view) DIC

612 Nematology

Pratylenchus arlingtoni n sp

P crenatus Loof 1960 P fallax Seinhorst 1968 P gibbi-caudatus Minagawa 1982 P gutierrezi Golden Loacutepez ampViacutelchez 1992 P pratensis (de Man 1880) Filipjev 1936P pseudofallaxCafeacute-Filho amp Huang 1989 P roseus Za-rina amp Maqbool 1998 P teres Khan amp Singh 1974 andP yassini Zeidan amp Geraert 1991

P crenatus P roseus and P teres may also have upto six lateral lines P arlingtoni n sp is morphologicallymost similar to P crenatus but differs by longer pharynx(93 - 107 vs 57 - 78 sup1m) pyriform to slightly overlappingbasal pharyngealglands (6 - 31 vs 17 - 38 sup1m) somewhatlower b value (41 - 53 vs 49 - 70 sup1m in P crenatusLoof 1960 Torres amp Chaves 1999 Urek 1999) smallerpost-uterine sac length relative to vulval-anal distance(PUSVA pound 100 28 - 42 vs 40 - 50) lower c0 value(11 - 15 vs 16 - 29) more elevated vulva and sixto eight lateral lines compared to four to six lines Theexcretory pore in P arlingtoni n sp is often below thepharyngeal-intestinal junction (p-ij) and is above thep-ij in P crenatus P arlingtoni n sp lacks a vulvalmembrane has a longer stylet (16 - 175 vs 15 - 16 sup1m)and a shorter pharyngealoverlap (6 - 31 vs 118 - 130 sup1m)compared to P roseus P arlingtoni n sp differs fromP teres by more posterior vulva (V = 81 - 86 vs 69 - 78)fewer tail annules (19 - 25 vs 24 - 30) shorter pharyngealoverlap (6 - 31 vs 58 sup1m) longer PUSVA pound 100 (28 - 42vs 18) and six to eight lateral lines rather than only six

When P arlingtoni n sp is compared to both P conva-llariae and P fallax it has a lower b value (41 - 53 vs 60- 90 and 52 - 67 respectively) and more posterior vulva(V = 81 - 86 vs 78 - 81 and 77 - 81 respectively) lacksmales and has two to four more lateral lines P arlingtonin sp also has more tail annules (19 - 25 vs 16 - 19) thanP convallariae The new species has a longer stylet (160 -175 vs 150 - 155 sup1m) more offset lip region and coarsertail annulation than P fallax Differences from P fallaxalso apply to P pseudofallax(Cafeacute-Filho amp Huang 1989)P arlingtoni n sp differs from P penetrans by having acrenate tail in all specimens lack of observedmales lowerb value (41 - 53 vs 53 - 79) shorter pharyngeal overlap(6 - 31 vs 32 - 65 sup1m) and longer PUSVW (15 - 25 vs10 - 15)

Observations on intercepted Pratylenchus fallaxand P convallariae

P fallax is common in sandy or sandy-peat soils (Sein-horst 1977) around grass and ornamentals of many Eu-ropean countries (Webb 1990) as well as the Canadian

provinces of Quebec and Ontario (Yu et al 1998) How-ever P fallax was found in the United States only in 1974on strawberries in Iowa (Norton 1984) Based on its geo-graphic distributionand pathogenicityto barley and maizein Europe (Corbett 1970 1972) it is underUS quarantinerestriction (Joseph Cavey pers comm)

A morphologicallyrelated species P convallariaeSein-horst 1959 is a non-endemic quality pest common inlight sandy soils (Seinhorst 1959) from Convallaria ma-jalis (lily of the valley) shipments from northern Euro-pean countries to the United States It was detected at leastsix times at the USDA Nematology Laboratory from late1998 to late 2000Because of its limited geographicdistri-bution but long-termpresence in the United States it is notsubject to regulatory action (Joseph Cavey pers comm)

P fallax was originally distinguished from P convalla-riae by its shorter body length narrower more numeroustail annules and lower male to female ratio (Seinhorst1968) P fallax was synonymisedby Frederick and Tarjan(1989) with P cerealis Haque 1965 because of the beliefshared by Loof (1978) that the attened condition of thetype specimens accounted for an arti cially much lowera ratio However the original description of P fallax alsodistinguished it from P cerealis by a longer body length(042 - 056 vs 039 - 043 mm) more posterior excretorypore (between nerve ring and pharyngeal-intestinal valvevs at level of median bulb in P cerealis) a longer tail andmore obscure atter lip annules (Seinhorst 1968)

Recently both P fallax and P convallariae were de-tected in a shipment of C majalis from France throughthe Netherlands and destined for the state of Ohio USAMountedspecimens of original paratypes of P fallaxwerecompared with P convallariae from a previous intercep-tion Intermediate morphologicalforms were also noticedbetween the typical P convallariae and P fallax Pho-tographs and supplemental measurements of representa-tive members of each species are provided and comparedwith previous descriptions

The P fallax population from France had typical spe-cies-diagnostic characters of crenate tail terminus in allmembers of the population (compared to P penetrans)oblique central lateral eld striations body length (048- 505 vs 050 - 056 mm in original description vs 058 -061 mm for P convallariae) rounded to anchor-shapedstylet knobs compared to more tulip-shaped knobs inP convallariae tail annule numbers (19 - 26 vs 16 - 19in P convallariae) and rare males to distinguish it fromP convallariae (Table 2 Fig 4) or P penetrans wheremales are more numerous Stylet lengths in this popula-

Vol 3(6) 2001 613

ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

Pratylenchus arlingtoni n sp

P crenatus Loof 1960 P fallax Seinhorst 1968 P gibbi-caudatus Minagawa 1982 P gutierrezi Golden Loacutepez ampViacutelchez 1992 P pratensis (de Man 1880) Filipjev 1936P pseudofallaxCafeacute-Filho amp Huang 1989 P roseus Za-rina amp Maqbool 1998 P teres Khan amp Singh 1974 andP yassini Zeidan amp Geraert 1991

P crenatus P roseus and P teres may also have upto six lateral lines P arlingtoni n sp is morphologicallymost similar to P crenatus but differs by longer pharynx(93 - 107 vs 57 - 78 sup1m) pyriform to slightly overlappingbasal pharyngealglands (6 - 31 vs 17 - 38 sup1m) somewhatlower b value (41 - 53 vs 49 - 70 sup1m in P crenatusLoof 1960 Torres amp Chaves 1999 Urek 1999) smallerpost-uterine sac length relative to vulval-anal distance(PUSVA pound 100 28 - 42 vs 40 - 50) lower c0 value(11 - 15 vs 16 - 29) more elevated vulva and sixto eight lateral lines compared to four to six lines Theexcretory pore in P arlingtoni n sp is often below thepharyngeal-intestinal junction (p-ij) and is above thep-ij in P crenatus P arlingtoni n sp lacks a vulvalmembrane has a longer stylet (16 - 175 vs 15 - 16 sup1m)and a shorter pharyngealoverlap (6 - 31 vs 118 - 130 sup1m)compared to P roseus P arlingtoni n sp differs fromP teres by more posterior vulva (V = 81 - 86 vs 69 - 78)fewer tail annules (19 - 25 vs 24 - 30) shorter pharyngealoverlap (6 - 31 vs 58 sup1m) longer PUSVA pound 100 (28 - 42vs 18) and six to eight lateral lines rather than only six

When P arlingtoni n sp is compared to both P conva-llariae and P fallax it has a lower b value (41 - 53 vs 60- 90 and 52 - 67 respectively) and more posterior vulva(V = 81 - 86 vs 78 - 81 and 77 - 81 respectively) lacksmales and has two to four more lateral lines P arlingtonin sp also has more tail annules (19 - 25 vs 16 - 19) thanP convallariae The new species has a longer stylet (160 -175 vs 150 - 155 sup1m) more offset lip region and coarsertail annulation than P fallax Differences from P fallaxalso apply to P pseudofallax(Cafeacute-Filho amp Huang 1989)P arlingtoni n sp differs from P penetrans by having acrenate tail in all specimens lack of observedmales lowerb value (41 - 53 vs 53 - 79) shorter pharyngeal overlap(6 - 31 vs 32 - 65 sup1m) and longer PUSVW (15 - 25 vs10 - 15)

Observations on intercepted Pratylenchus fallaxand P convallariae

P fallax is common in sandy or sandy-peat soils (Sein-horst 1977) around grass and ornamentals of many Eu-ropean countries (Webb 1990) as well as the Canadian

provinces of Quebec and Ontario (Yu et al 1998) How-ever P fallax was found in the United States only in 1974on strawberries in Iowa (Norton 1984) Based on its geo-graphic distributionand pathogenicityto barley and maizein Europe (Corbett 1970 1972) it is underUS quarantinerestriction (Joseph Cavey pers comm)

A morphologicallyrelated species P convallariaeSein-horst 1959 is a non-endemic quality pest common inlight sandy soils (Seinhorst 1959) from Convallaria ma-jalis (lily of the valley) shipments from northern Euro-pean countries to the United States It was detected at leastsix times at the USDA Nematology Laboratory from late1998 to late 2000Because of its limited geographicdistri-bution but long-termpresence in the United States it is notsubject to regulatory action (Joseph Cavey pers comm)

P fallax was originally distinguished from P convalla-riae by its shorter body length narrower more numeroustail annules and lower male to female ratio (Seinhorst1968) P fallax was synonymisedby Frederick and Tarjan(1989) with P cerealis Haque 1965 because of the beliefshared by Loof (1978) that the attened condition of thetype specimens accounted for an arti cially much lowera ratio However the original description of P fallax alsodistinguished it from P cerealis by a longer body length(042 - 056 vs 039 - 043 mm) more posterior excretorypore (between nerve ring and pharyngeal-intestinal valvevs at level of median bulb in P cerealis) a longer tail andmore obscure atter lip annules (Seinhorst 1968)

Recently both P fallax and P convallariae were de-tected in a shipment of C majalis from France throughthe Netherlands and destined for the state of Ohio USAMountedspecimens of original paratypes of P fallaxwerecompared with P convallariae from a previous intercep-tion Intermediate morphologicalforms were also noticedbetween the typical P convallariae and P fallax Pho-tographs and supplemental measurements of representa-tive members of each species are provided and comparedwith previous descriptions

The P fallax population from France had typical spe-cies-diagnostic characters of crenate tail terminus in allmembers of the population (compared to P penetrans)oblique central lateral eld striations body length (048- 505 vs 050 - 056 mm in original description vs 058 -061 mm for P convallariae) rounded to anchor-shapedstylet knobs compared to more tulip-shaped knobs inP convallariae tail annule numbers (19 - 26 vs 16 - 19in P convallariae) and rare males to distinguish it fromP convallariae (Table 2 Fig 4) or P penetrans wheremales are more numerous Stylet lengths in this popula-

Vol 3(6) 2001 613

ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

ZA Handoo et al

Table 2 Morphometrics of Pratylenchus fallax and P convallariae (after Seinhorst 19591 19682 19773 Loof 19914 curren D measurederived from original description) Values are in sup1m and those followed by (n D 10) were derived by the authors and are presentedas range followed by mean sect standard deviation

Measure P fallax Seinhorst 1968 P convallariae Seinhorst 1959

L 420 - 560 580 - 610a 24 - 33 23 - 27b 52 - 67 60 - 90c 18 - 24 17 - 28V 77 - 81 78 - 81Stylet length 15 - 155 16 - 17Tail annule number 16 - 26 16 - 19Lateral line number 4 centre often oblique 4Post-uterine sacVulval width 10 - 16 14 - 20Post-uterine sacVulva-anus pound 100 25 - 33 21 - 25Pharyngeal overlap 20 - 44 (31)4 31 - 55 (41)4

MaleFemale ratio 1 5 up to 1 1Spermatheca shape roundoval when empty roundLip number 3 3 offsetExcretory pore position atposterior to nerve ring atposterior to nerve ringPhasmid-tail terminus 9 - 13 annules3 6 - 8 annules (n D 10)Pharynx 92 - 115 1026 sect 77 (n D 10) 102 - 122 111 sect 72 (n D 10)Pharyngeal overlap 25 - 35 302 sect 37 (n D 10) 30 - 40 341 sect 31 (n D 10)b0 43 - 52 48 sect 04 (n D 10) 40 - 48 44 sect 02 (n D 10)c0 17 - 22 20 sect 02 (n D 10) 20 - 24 22 sect 01 (n D 10)Post-uterine sac 16 - 25 206 sect 34 (n D 10) 16 - 26 199 sect 26 (n D 10)Vulva-anus distance 72 - 90 788 sect 61 (n D 10) 70 - 88 768 sect 65 (n D 10)Vulval width 16 - 20 179 sect 13 (n D 10) 14 - 20 171 sect 16 (n D 10)Vulva-spermatheca 28 - 42 326 sect 49 (n D 10) 23 - 50 342 sect 73 (n D 10)Vulva-spermathecaVulva-anus pound 100 31 - 58 42 sect 10 (n D 10) 33 - 59 44 sect 10 (n D 10)

tion (155 - 17 sup1m) overlapped the narrow range for bothP fallax (15 - 155 sup1m) and P convallariae (16 - 17 sup1m)The P fallax population also had male tails with a slightconcavity on the posterior third of the bursa (Fig 4I) as inthe original drawing (Seinhorst 1968) This P fallax malebursal pro le with the concavity after the papillar phas-mid is different from the uniformly convex bursal pro- le descriptions (Seinhorst 1959) and observations fromP convallariae (Fig 4N) Measurement ranges (Table 2)supplementing those in the literature include c0 and b0

values post-uterine sac length vulva-anusdistance vulvawidth vulva spermatheca and vulva-spermatheca relativeto vulva-anus distance These measures are similar forP fallax and P convallariae but pharynx lengthor overlapwas slightly greater in P convallariae than P fallax Thepharyngeal overlaps occurred within the range of thosealready published (Loof 1991) However even the high-est value of the 30 - 40 sup1m overlap (average = 34 sup1m)measured here for P convallariae fell within the lower

quarter of the 32 - 65 sup1m range for P penetrans overlap(Loof 1991) The number of tail annules from the termi-nus to the phasmid was another differentiating characterbetween P fallax (9 - 13 annules) and P convallariae(lessthan nine annules) based on literature descriptions andobservations in specimens from these two populationsExceptional individuals had P fallax-like tail annulationwith P convallariae-like stylet knob-shape and length orbifurcated tail termini with more tail annules One ofthese individuals had identical 28S rDNA D3 sequencesto those of completely true-to-type P convallariae andP fallax from separate shipments Conversely we mea-sured a population (n = 10) with all the other characteris-tics of P convallariaeexcept for smaller body length (046- 058 mm) that bridges the originally non-overlappingpublished length ranges for P fallax (042 - 056 mm)(Seinhorst 1959) and P convallariae (058 - 051 mm)(Seinhorst 1968) A voucher specimen (UCDNC 3279)of this P crenatus conformed to the species description

614 Nematology

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

Pratylenchus arlingtoni n sp

Fig 4 Comparison of Pratylenchus fallax and P convallariae (lateral view) DIC A B P fallax head C P fallax lateral eld midbodyD P fallax female tail E P fallax male tail F G P convallariae female heads H P convallariae lateral eld mid-body I JP convallariae female tails

Molecular characterisation

Shown in Fig 5 are aligned sequences of the D3 regionof the LS 28S rDNA for P arlingtoni designated as lsquoarlrsquofor P convallariae as lsquoconrsquo and for P fallax as lsquofalrsquoAlso shown is the sequence for P penetrans (Al-Bannaet al 1997) from the GenBank database that most nearlyresembled our new sequences

The 28S rDNA sequences of typical specimens ofP convallariae and P fallax were identical P arling-toni n sp differed from that sequence by three base pairs(bp)305 (1 difference) which included a G substitu-tion from A at position 55 in the sequence or 56 in thealignment and a T substitution from C at position 71 in

the sequence or 73 in the alignment both within the vari-able region of the sequence The third change a G inser-tion between G and T at position 251 in the sequence or256 in the alignment occurred in a highly conserved re-gion of the molecule The P arlingtoni sequence differedby 9305 bp (3 difference) from P penetrans which in-cluded the previous changes plus changes in commonwith P fallax and P convallariae These shared differ-ences among the three species relative to P penetrans in-cluded a C substitution from G at position 39 in the align-ment C substitution from A at position 69 G from T atposition 71 T from A at position 82 G from A at po-sition 84 C from T at position 139 G from A at posi-tion 148 and A from G at position 231 The sequence

Vol 3(6) 2001 615

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

ZA Handoo et al

Fig 5 Sequence alignments of LS 28SrDNA with Clustal W for Pratylenchusarlingtonin sp P convallariaeP fallax and P penetranspen Closest sequence in Genbank to the others P penetrans accessionnumber U47546 (Al-Banna et al 1997) arl P arlingtoni conP convallariae fal P fallax ndash Gap Nucleotides differing from the others are underlined and in bold

of P fallaxP convallariae differed from P penetrans by12305 bp (4) The sequence for P crenatus Accessionnumber U47549 (not shown in Table 5) (Al-Banna et al1997) was fairly distant from P arlingtoni n sp differ-ing by 49305 bp (16) where all but six major changesoccurred in a variable region between alignment positions60 and 175

Discussion

The unusual character of six or eight lateral lines wasreported (Roman amp Hirschmann 1969) in a small pro-portion of the populations of P brachyurus (Godfrey1929) Filipjev amp Schuurmans Stekhoven1941 P coffeae(Zimmerman 1898) Filipjev amp Schuurmans Stekhoven1941 P penetrans (Cobb 1917) Filipjev amp SchuurmansStekhoven 1941 P scribneri Steiner 1943 P vulnusAllen amp Jensen 1951 and P zeae Graham 1951 How-

ever P arlingtoni n sp is the rst nematode with a con-sistent six to eight just as four to six have been found in afew other lesion nematode species

Pharyngeal characters of P arlingtoni n sp are im-portant for its identi cation The pharyngeal overlap wasa diagnostically and phylogenetically reliable characterin well-preserved specimens in recent morphological andmolecular studies with multiple Pratylenchus species(Loof 1991 Al-Banna et al 1997 Duncan et al 1999)

Despite the morphological similarity of P arlingtonin sp to P crenatus populations described from Europe(Loof 1960 1991 Urek 1999) and South America (Tor-res amp Chaves 1999) there is substantial 28S rDNA se-quence difference between P arlingtoni n sp and theP crenatus population from Oregon (Al Banna et al1997) and a population from Ohio (unpubl) Just as Al-Banna et al (1997) proposed the genus Pratylenchus tobe polyphyletic crenate-tailed nematodes with P crena-tus-like morphology might be polyphyleticas well

616 Nematology

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

Pratylenchus arlingtoni n sp

Although P arlingtoni n sp and P crenatus are mor-phologically similar they are molecularly distant How-ever the identical DNA sequences here for P fallax andP convallariaecould lend support to a proposal that P fal-lax be considered a subspecies or synonym of P conval-lariae due to morphologicalsimilarity It would be prema-ture to synonymise P fallax as P cerealis as suggested byFrederick and Tarjan (1989) when both species might bebetter synonymised with P convallariae due to overlap-ping morphological and molecular characters and prior-ity of descriptionBreeding studies might be helpful sincethe morphologicallyintermediate specimens in this reportmay have been sterile hybrids of two species or simplynatural variants within a single biological species

P fallax was originally described as being morpholog-ically similar to the highly variable P penetrans (Sein-horst 1968) with fewer males a generally longer pharyn-geal overlap (Loof 1991) and occasionalpopulationswithcrenate tails (Tarteacute amp Mai 1976) P fallax was also dif-ferent from P penetrans in lacking fertile hybrid progeny(Perry et al 1980) and on isozyme gels (Ibrahim et al1995) or with restriction-enzyme-digested fragments ofthe rDNA ITS region (Waeyenberge et al 2000) Here itappears that P fallax may share at least as close a biologi-cal relationship with P convallariae as with P penetransBoth P convallariae and P fallax have relatively narrowoverlapping morphometric ranges compared to other le-sion nematodes based on keys and information presentedhere and both are found with similar soil types hosts andoverlapping geographic regions However careful com-parative pathogenicity testing has been limited for P con-vallariae and P fallax (Webb 1990)

Because of the morphological and molecular relation-ship of P arlingtoni n sp P fallax and P convallar-iae and their limited distribution or absence in the UnitedStates there is a need for further survey of lesion nema-todes particularly in the United States Canada and Eura-sia It is dif cult to believe that P fallax is restricted to thesouthern provinces of Canada but not the northern statesof the United States However if true there may be a par-allel in P fallax being common in Great Britain (Corbett1970) while both P convallariae and P fallax are foundat lower latitudes in Europe (Seinhorst 1959 1977) Thein uence of temperature on morphological variability inthese species as currently characterised might be consid-ered Further tests of European Canadian and US popu-lations of these related nematodes are needed to evaluatecurrent regulatory restrictions on P fallax

Acknowledgements

The authors thank Donna Ellington Roberta Henegarand Sharon Ochs for technical assistance and JosephCavey and Michael Firko Animal and Plant Health In-spection Service USDA Riverdale MD USA for reg-ulatory information The authors wish to thank GerritKarssen Plant ProtectionService WageningenThe Neth-erlands for sharing information on P fallax and P conva-llariae The authors thank Pierre Baujard Gerrit Karssenand Michel Luc for manuscript review Mention of tradenames or commercial products in this publication is solelyfor the purpose of providing speci c informationand doesnot imply recommendation or endorsement by the UnitedStates Department of Agriculture

References

AL-BANNA L W ILL IAMSON V amp GARDNER SL (1997)Phylogenetic analysis of nematodes of the genus Praty-lenchus using nuclear 26S rDNA Molecular Phylogeneticsand Evolution 7 94-102

BALDWIN JG FRISSE LM VIDA JT EDDLEMANCD amp THOMAS WK (1997) An evolutionary frameworkfor the study of developmentalevolution in a set of nematodesrelated to Caenorhabditis elegans Molecular Phylogeneticsand Evolution 8 249-259

CAFEacute-FILHO AC amp HUANG CS (1989) Description ofPratylenchus pseudofallax n sp with a key to species of thegenus Pratylenchus Filipjev 1936 (Nematoda Pratylenchi-dae) Revue de Neacutematologie 12 7-15

CHOU Q RUSSELL M BIRCH DE RAYMOND J ampBLOCH W (1992) Prevention of pre-PCR mis-priming andprimer dimerization improves low-copy-number ampli ca-tions Nucleic Acids Research 20 1717-1723

CORBETT DCM (1970)Cereal Pratylenchusspp in Englandand Wales and their recognition Plant Pathology 19 6-10

CORBETT DCM (1972) The effect of Pratylenchus fallax onwheat barley and sugar beet roots Nematologica 18 303-308

DUNCAN LW INSERRA RN THOMAS WK DUNN DMUSTIKA I FRISSE LM MENDES ML MORRIS K amp KAPLAN DT (1999) Genetic and morphologicalrelationships among isolates of Pratylenchus coffeae andclosely related species Nematropica 29 61-80

ELLIS RE SULSTON JE amp COULSON AR (1986) TherDNA of C elegans sequence and structure Nucleic AcidsResearch 14 2345-2364

FIL IPJEV IN (1936) On the classi cation of the TylenchinaeProceedings of the Helminthological Society of Washington3 80-82

Vol 3(6) 2001 617

ZA Handoo et al

FREDERICK JJ amp TARJAN AC (1989) A compendium ofthe genus Pratylenchus Filipjev 1936 (Nemata Pratylenchi-dae) Revue de Neacutematologie 12 243-256

GOLDEN AM (1990) Preparation and mounting nematodesfor microscopic observation In Zuckerman BM Mai WFamp Krusberg LR (Eds) Plant nematology laboratory man-ual Revised Edition Amherst MA USA University ofMassachusetts Agricultural Experimental Station pp 200-201

GOLDEN AM LOPEZ CHR amp VILCHEZ RH (1992)De-scription of Pratylenchus gutierrezi n sp (Nematoda Praty-lenchidae) from coffee in Costa Rica Journal of Nematology24 298-304

HAQUE MM (1965) Two new species of plant nematodesDoklady Timiryazevskoi Selskokhozyaistrennoi Akademii113 209-219

IBRAHIM SK PERRY RN amp WEBB RM (1995) Useof isoenzyme and protein phenotypes Annals of AppliedBiology 126 317-327

KHAN E amp SINGH DB (1974) Five new species of Praty-lenchus (Nematoda Pratylenchidae) from India Indian Jour-nal of Nematology 4 199-211

LOOF PAA (1960) Taxonomic studies on the genus Praty-lenchus (Nematoda) Tijdschrift voor Plantenziekten 66 29-90

LOOF PAA (1978) The genus Pratylenchus Filipjev 1936(Nematoda Pratylenchidae) a review of its anatomy mor-phology distributionsystematics and identi cation Vaxtsky-ddsrapporter 5 50 pp

LOOF PAA (1991) The family PratylenchidaeThorne 1949In Nickle WR (Ed) Manual of Agricultural NematologyNew York NY USA Marcel Dekker Inc pp 363-421

MINAGAWA N (1982) Descriptionsof Pratylenchusgibbicau-datus n sp and P macrostylus Wu 1971 (Tylenchida Praty-lenchidae) from Kyushu Japanese Journal of Applied Ento-mology and Zoology 17 418-423

NORTON DC (1984) Distribution of plant-parasitic nema-tode species in North America Hyattsville MD USA So-ciety of Nematologists 199 pp

PERRY RN PLOWRIGHT RA amp WEBB RM (1980)Mating between Pratylenchus penetrans and P fallax insterile culture Nematologica 26 125-129

ROMAN J amp HIRSCHMANN H (1969) Morphology andmorphometrics of six species of Pratylenchus Journal ofNematology 1 363-386

SEINHORST JW (1959) Two new species of PratylenchusNematologica 4 83-86

SEINHORST JW (1968) Three new Pratylenchus species witha discussion of the structure of the cephalic framework and ofthe spermatheca in this genus Nematologica 14 497-510

SEINHORST JW (1977) Pratylenchusfallax CIH descriptionsof plant parasitic nematodes St Albans UK CommonwealthAgricultural Bureaux Set 7 No 98 pp 1-2

STIERNAGLE T (1999) Maintenance of C elegans In HopeIA (Ed) C elegans a practical approach New York NYUSA Oxford University Press pp 51-67

TARTEacute R amp MAI WF (1976) Morphological variation inPratylenchus penetrans Journal of Nematology 8 185-195

THOMPSON JD HIGGINS DG amp GIBSON TJ (1994)CLUSTAL W improving the sensitivity of progressivemultiple sequence alignment through sequence weightingposition-specic gap penalties and weight matrix choice Nu-cleic Acids Research 22 4673-4680

TORRES MS amp CHAVES EJ (1999) Description ofsome Pratylenchidae (Nemata) from Argentina NematologiaMediterranea 27 281-289

UREK G (1999) The effect of host plants on the biopotentialand the morphometric characteristics of Pratylenchus crena-tus Loof 1960 Acta Phytopathologicaet Entomologica Hun-garica 34 253-262

WAEYENBERGE L RYSS A MOENS M PINOCHET Jamp VRAIN TC (2000) Molecular characterisation of 18Pratylenchus species using rDNA restriction fragment lengthpolymorphism Nematology 2 135-142

WEBB RM (1990) Effects of the nematode Pratylenchusfallax on roots of oilseed rape (Brassica napus var oleifera)Revue de Neacutematologie 13 115-117

W ILL IAMS BD SCHRANK B HUYNH C SHOWNKEENR amp WATERSTON RH (1992) A genetic mapping systemin Caenorhabiditis elegans based on polymorphic sequence-tagged sites Genetics 131 609-624

YU Q POTTER JW amp GILBY G (1998) Plant parasitic ne-matodes associated with turfgrass in golf courses in SouthernOntario Canadian Journal of Plant Pathology 20 304-307

ZARINA B amp MAQBOOL MA (1998) Descriptions andobservations on two new and two known species of the genusPratylenchusFilipjev1936 (Nematoda Pratylenchidae)fromPakistan Pakistan Journal of Nematology 16 13-24

ZEIDAN AB amp GERAERT E (1991) Pratylenchus fromSudan with the description of two new species (NemataTylenchida) Revue de Neacutematologie 14 261-275

618 Nematology