Nematology,2001,V ol.3(6), 607-618

Morphological andmolecular characterisation of Pratylenchus arlingtoni n. sp., P.convallariae and P. fallax (Nematoda: Pratylenchidae)

Zafar A. HANDOO, Lynn K. CARTA ¤ andAndrea M. S KANTAR UnitedStates Department of Agriculture,ARS, NematologyLaboratory, Beltsville, MD 20705,USA Received:17 October2000; revised: 19 June2001 Acceptedfor publication:23 July2001

Summary – Pratylenchusarlingtoni n.sp. from the rhizosphere of grasses Poapratensis and Festucaarundinacea atArlington NationalCemetery, V A,USA ischaracterised by sixto eight lines in thelateral Ž eld,and pyriform to slightlyoverlapping pharyngeal glands.Morphological comparisons are made with lesion nematodes having similar morphometrics, six lateral lines, or crenate tail tips. Molecularsequences of theLS 28SrDNA weregenerated for the new species as wellas P. fallax and P.convallariae .Thenew species differsby only 1% fromidentical sequences found in P. fallax and P.convallariae . Keywords – Festucaarundinacea ,lesionnematode, pathogenicity, Poapratensis , Pratylenchuscrenatus ,quarantine.

Duringa searchfor nematodesin Arlington National P.convallariae and P. fallax were isolatedfrom inter- Cemetery,Arlington, V A,USA, we discovereda new ceptedlily of thevalley shipments (March, 1999 and Jan- lesionnematode from soilaround grass roots. This new uary,2000, respectively, from acompanyin Europe) af- speciesdescribed hereunder as Pratylenchusarlingtoni tercollection at JFK InternationalAirport, Jamaica, New n.sp. had some morphological similarities to crenate- Yorkby BerniceMedina and dispatched to us,for identiŽ- tailed Pratylenchuscrenatus Loof,1960 and Pratylenchus cation,by AlanT owson,USDA, Animaland Plant Health fallax Seinhorst,1968, two other lesion nematode species InspectionService (APHIS). foundin turf in North America and Europe (Y u et al., Nematodeswere extractedfrom soilwith sieves or from 1998). chopped,infested roots over Ž lterpaper in Baermann Thepurpose of thisstudy was tomorphologicallyand funnels.Specimens were thenhandpicked and Ž xedin molecularlydescribe the new nematode and compare it 3%formalin. Some specimens were studiedin Ž xative withnematodes having similar characters, such as P. fal- ontemporary slide mounts, others in permanent glycer- lax and P.convallariae Seinhorst,1959. One of these inemounts (Golden, 1990), or viewedlive, with or with- characterswas theD3 region of the 28S rDNA, which out5 mMsodiumazide for narcotisationon anagarpad encodespart of the Large Subunit (LS) rRNA (Baldwin (Stiernagle,1999). Examinations were madewith a com- et al.,1997)recently used to characterise other species poundlight microscope and morphometric data obtained of Pratylenchus andtheir phylogenetic relationships (Al- withan ocular micrometer. All measurements are in mi- Banna et al., 1997;Duncan et al., 1999). crometers (¹m) unlessotherwise speciŽ ed. Light micro- scopicimages of live and Ž xednematodes were taken Materials andmethods withthe Bioquant ver .3.2imaging system (Biometrics, Inc.,Nashville, TN, USA) ona LeitzOrtholux micro- scope.Differential interference contrast (DIC) imagesof MORPHOLOGICALCHARACTERISATION livenematodes were takenwith an imaging system em- P.arlingtoni n.sp. was foundin turf ( Poapratensis ployingImage-Pro Plus ver 3.0 (I-Cube Image Analy- and Festucaarundinacea )soilfrom ArlingtonNational sis/ImageProcessing, Crofton, MD, USA) onaZeissUl- Cemetery,Arlington, V A,USA, inNovember, 1999. traphotII microscopeequipped with DIC optics.Nema-

¤ Correspondingauthor, e-mail: [email protected] c KoninklijkeBrill NV ,Leiden,2001 607 ° Z.A. Handoo et al. todeswere processedfor scanningelectron microscopy todeextract was includedin each PCR reaction.The re- (SEM) inroom temperature 0.05 phosphate-buffered (pH sultantPCR productswere clonedinto the vector pCR2.1 6.8)3% glutaraldehyde(12 h) and2% osmiumtetroxide usingthe T opo-TACloningkit (Invitrogen,Carlsbad, CA, (2h) and viewed after ethanol dehydration, critical-point USA). PlasmidDNA was puriŽed from bacterialcultures dryingand gold-palladium coating on a JeolJSM-T300 usingWizard Preps (Promega, Madison, WI, USA). The microscopeat 20 kV. sequencingreactions contained 200 ng plasmid template andthe M13 forward orM13 reverse primers. All BigDye TEMPLATEPRE PARATION Terminatorcycle sequencing was performedusing an ABI 377Sequencer (PE-Applied Biosystems, Foster City, CA, Nematodeextracts were preparedby the procedure USA). of Williams et al.(1992).A singlenematode was pla- ced in 10 ¹lofdigestion buffer (10 mM Tris pH 8.2; Negativecontrols included reactions with water or a 2.5 mM MgCl 50mM KCl; 0.45% T ween20; 0.05% mockextract (no nematode) instead of DNA. Areac- 2I gelatin; 60 ¹g/mlproteinase K) andfrozen at 70 C for tioncontaining 5 ng Meloidogynejavanica genomic DNA ¡ ± 15min to several days. The extracts were thawed,over- was includedas a positivecontrol. T oconŽrm theau- thenticityof the sequences obtained, PCR ampliŽcation laidwith a dropof mineraloil, and warmed to 60 ±C for 1h.Proteinase K was denaturedby heating to 95 ±C for andDNA sequencingwere performedon two individu- 15 min. alsfrom thesame nematode population. T oaccountfor thepossibilityof PCR-generatederrors inthe clonedPCR AMPLIFICATIONANDSEQUENCING products,we comparedthe sequences from twoor more clonesobtained from thesame nematode extract. If ambi- TheD3expansion region of 28SrDNA (345nucleotide guitieswere detectedbetween clones, sequences derived basepairs (bp) raw sequence)was ampliŽed separately directlyfrom PCRproducts were usedto resolvethe con- from twoadult nematodes of each species, using hot-  ict. startreactions as describedby Chou et al.(1992)with the Thesequences for P.arlingtoni n. sp., P.convallariae followingmodiŽ cations. Manufacturer-supplied Display- and P. fallax havebeen deposited in the GenBank data- TAQ buffer(PGC ScientiŽc, Gaithersburg, MD, USA), base(National Center for BiotechnologyInformation, Na- 250 ¹MdNTPs,4 mMMgCl , and 600 ¹M of each ri- 2 tionalLibrary of Medicine, National Institute of Health, bosomalDNA primer,originally designed by W .Kelley Bethesda,MD, USA, http://www.ncbi.nlm.nih.gov)as Thomas,University of Missouri,Kansas City, MO, USA. AF307328,AF196351, and AF264181, respectively. Primers D3A (5 0-GACCCGTCTTGAAACACGGA-3 0) and D3B (50-TCGGAAGGAACCAGCTACTA-3 0) (Bald- win et al.,1997)were addedto the bottom of 0.5ml thin- wallmicrocentrifuge tubes. A drop( ca 25 ¹l) of paraf- ALIGNMENT Žnwaxwas overlaidand allowed to cool, forming an evenbarrier. The remaining T AQbuffer,template and Dis- From the345 bp of raw sequence,305 bp were used playT AQwere thenlayered on top of the wax. Cycling inthe Ž nalalignment. The new sequences were aligned conditionswere: 94 C,3min(to allow hot start); 94 C, ± ± withthe Clustal W (ver 1.4)program (Clustal W ,WWW 1 min; 52 C, 1 min; 72 C, 1 min, 35cycles; 72 C, ± ± £ ± Serviceat the European Bioinformatics Institute, Ro- 10min. Reactions were analysedby gel electrophore- drigoLopez, Services Programme, http:/ /www2.ebi.ac. sis. DNA sequenceswere obtainedby sequencing PCR uk/clustalw;Thompson et al.,1994)with all Pratylenchus productsdirectly or bysequencingcloned PCR products. sequencesin the GenBankdatabase. The closest sequence For directsequencing, whole nematode extracts (10 ¹l) tothe new species from theGenBank database is also were includedin thePCR reactions to generate a sufŽcient shownin the alignment provided here (Fig. 5). Positions amountof PCR product.Prior tosequencing, the DNA arenumbered where 1 correspondsto number 3324 of was puriŽed usingthe Qiaquick PCR puriŽcation kit (Qi- the Caenorhabditiselegans 28SrRNA gene(Ellis et al., agen,V alencia,CA, USA). Sequencingreactions included 1986;Al-Banna et al.,1997).The number and posi- 100ngPCR productand 3.2 pmolD3A orD3B primer. T o tionof nucleotide differences among the four taxa were generatecloned PCR productsfor sequencing,2 ¹l nema- noted.

608 Nematology Pratylenchusarlingtoni n. sp.

Table 1. Morphometricsof Pratylenchusarlingtoni n.sp. females. (All measurements in ¹m.)V aluesare means standarddeviation. § Valuesin parentheses indicate ranges. n 20 Holotype Paratypes D L 423 455 37 (405 - 535) § a 24.8 27 3 (21 - 33) § b 4.0 4:4 0:4 (4.1 - 5.3) § c 20.6 22 2 (18 - 28) § V 81.5 82:3 1:2 (81 - 86) § Styletlength 17 16:8 0:5 (16 - 17.5) § c 2.0 1:3 0:1 (1.1 - 1.5) 0 § Tailannule number 21 20:9 1:8 (19 - 25) § Lateralline number – – (6- 8)aerolate extremes Post-uterinesac 25 19:4 2:8 (15 - 25) § Vulvalwidth 15 15:9 1:1 (14 - 17.5) § Vulva-Anusdistance 53 55:9 7:1 (43 - 70) § Post-uterinesac/ Vulvalwidth 1.7 – (1.5 - 2.5) Post-uterinesac/ Vulva-anus 100 47 – (28 - 42) £ Pharyngealoverlap – 20 7:8 (6- 31)pyriform to overlap § Males/spermatheca – Notfound/ oval Lipnumber, shape – 3,slightlyoffset Excretorypore position – Pharyngeal-intestinaljunction Excretorypore-lips – 70 10 (48 - 85) § Body diam. 15 17 1:4 (15 - 20) § Pharynxlength – 101 3:5 (93 - 107) § Tail length – 21 1:8 (15 - 23) § Styletknob height – 2:7 0:3 (2.5 - 3.0) § Styletknob width – 4:6 0:2 (4.5 - 5.0) § Styletknob width/ height – 1:7 0:1 (1.6 - 1.8) § Head diam. – 8:1 0:4 (7.5 - 9.0) C Head height – 2:8 0:3 (2.5 - 3.0) § Headdiam./ height – 2:8 0:3 (2.6 - 3.2) §

Pratylenchus arlingtoni ¤ n. sp. directedslightly forward. Dorsalpharyngeal gland open- (Figs 1-3,5) ing at 2-3 ¹mbehindthe stylet knobs. Lateral Ž eldbegin- ningbehind the level of thestylet as fournarrow crenate MEASUREMENTS lines,widening to Ž veby the level of the median bulb, andsix to eight by the anterior intestine level through to Females thevulva. Four lines extending to the phasmid just pos- See Table 1. teriorto the vulva, after which three lines extend a few annulesshort of thetail tip. Lateral Ž eldsometimes aero- DESCRIPTION latedat the extremes. Pharyngeal glands pyriform in ap- Female proximately25% ofthespecimensor slightlyoverlapping inothers.Excretory pore and canal located within an area Bodyvermiform with some tapering at extremities. Lip regionslightly offset, three annules. Head framework ex- slightlyabove or belowthe pharyngeal-intestin aljunction. tendinginward for twoor threeannules. Anterior body an- Vulvaelevated, the slit extending inward about 70% ofthe nulesmeasuring 1.2 ¹m,tail annules measuring 0.9 ¹m. bodydiam. Anterior gonad with a singlerow ofoocytes Styletknobs broadly rounded, with anterior outer edges andextending forward for nearlythree times the distance betweenthe vulva and anus. An egg (36-cell) within the * FromArlington, V A,USA, typelocality. bodymeasuring 75 21 ¹m;another (4-cell) 64 14 ¹m. £ £ Vol.3(6), 2001 609 Z.A. Handoo et al.

Ovalspermatheca, 17 ¹minlengthwith apparent sperm cellsof 1.4- 2.2 ¹m diam., 74 ¹manteriorto the vulva (onespecimen out of at least 200). Distance from vulva tospermatheca, 120% of thedistance between vulva and anus.Post-uterine sac generally undifferentiated, with a discernibletip cell or four columnar cells at the dorsal archsometimes present. Phasmid located 10 to15 annules abovethe tail tip. T ailterminus coarsely annulated, with variableshape from conoid,clavate to truncate,or some- times biŽ d.

Male Not found.

TYPEHOSTANDLOCALITY

Rootsof turf ( Poapratensis ,bluegrass and Festuca arundinacea ,tallfescue) under Quercus spp.(oaks) at OldSection 27, ArlingtonCemetery, Arlington, V A,USA. Thissection of thecemetery was from theold Arlington Farm, uncultivatedsince the 1800s.

TYPESPECIMENS

Holotype SlideT -543pdeposited in the United States Depart- mentof Agriculture Nematode Collection (USDANC), Beltsville,MD, USA.

Paratypes(females) Samedata and repository as holotype.Slides T -4918p- 4921pwith 15 mixed stage females and slides T -4922p- 4924pwith single adult females. Others deposited in thenematode collections at The University of Califor- niaDavis, Davis, CA, USA; TheCanadian Food Inspec- tionAgency, Centre for PlantQuarantine Pests, Ontario, Canada;Rothamsted Experimental Station, Rothamsted, UK; Muséum Nationald’ Histoire Naturelle, Laboratoire desV ers,Paris, France; W ageningenUniversity and Re- searchCentre, Landbouwhogesch ool,W ageningen,The Netherlands,and the Zoological Institute, Russian Acad- emyof Sciences,St Petersburg, Russia. Fig. 1. Pratylenchusarlingtoni n.sp. (lateral views) A: Body;B: LateralŽ eld,midbody; C: Headand pharynx; D -F:Tail. DIAGNOSISANDRELATIONSHIPS

P.arlingtoni n.sp. is unique among lesion nematode alsohas a crenatetail tip, an elevated vulva, no observed speciesin having six to eight lateral lines on specimens males,and a veryrare, ovalspermatheca. from thepharyngeal to the vulval region, and pharyngeal P.arlingtoni n.sp. shares a variablycrenate tail with glandswith a pyriformbasal bulb to a shallowoverlap. It P. cerealis Haque,1965, P.convallariae Seinhorst,1959,

610 Nematology Pratylenchusarlingtoni n. sp.

Fig. 2. Pratylenchusarlingtoni n.sp. Scanning electron micrographs. A, B:Lipregion (face view) with and without amphid secretion; C:Lipregion (lateral view); D: Body,E: Tailand phasmid opening (arrow); F: LateralŽ eld;G: Tailregion (lateral view).

Vol.3(6), 2001 611 Z.A. Handoo et al.

Fig. 3. Pratylenchusarlingtoni n.sp.A: Headend (lateral) with median bulb, light microscopy (LM); B: Pharynx,pyriform basal bulb (ventrolateralview), LM; C:Femaletail (lateral view), LM; D:Head(lateral view), Differential interference contrast (DIC); E: Lateral Želd,midbody LM; F:Pharyngeal-intestinaljunction (pij) and excretory canal (exc) (lateral view), DIC; G: LateralŽ eld,mid-body, DIC;H: Spermathecawith sperm (lateral view), DIC; I: Femalegonad with vulva, egg, post-uterine sac (lateral view), DIC.

612 Nematology Pratylenchusarlingtoni n. sp.

P. crenatus Loof, 1960, P. fallax Seinhorst,1968, P. gibbi- provincesof Quebecand Ontario (Y u et al.,1998).How- caudatus Minagawa,1982, P.gutierrezi Golden,Ló pez & ever, P. fallax was foundin the United States only in 1974 Vílchez, 1992, P.pratensis (deMan, 1880) Filipjev,1936, onstrawberriesin Iowa (Norton,1984). Based on itsgeo- P.pseudofallax Café-Filho & Huang,1989, P. roseus Za- graphicdistribution and pathogenicity to barley and maize rina& Maqbool,1998, P. teres Khan& Singh,1974, and inEurope(Corbett, 1970, 1972),it is under USquarantine P. yassini Zeidan& Geraert,1991. restriction(Joseph Cavey, pers. comm.). P.crenatus,P .roseus and P. teres mayalso have up Amorphologicallyrelated species, P.convallariae Sein- tosix lateral lines. P.arlingtoni n.sp.is morphologically horst,1959 is a non-endemicquality pest, common in mostsimilar to P.crenatus, butdiffers bylongerpharynx light,sandy soils (Seinhorst, 1959) from Convallariama- (93 - 107 vs 57 - 78 ¹m), pyriformto slightly overlapping jalis (lilyof the valley) shipments from northernEuro- basalpharyngeal glands (6 - 31 vs 17 - 38 ¹m), somewhat peancountries to theUnitedStates. It was detectedat least lowerb value(4.1 - 5.3 vs 4.9 - 7.0 ¹m in P. crenatus; sixtimes at theUSDA NematologyLaboratory from late Loof,1960; T orres &Chaves,1999; Urek, 1999), smaller 1998tolate 2000. Because of itslimited geographic distri- post-uterinesac length relative to vulval-anal distance butionbut long-term presence in theUnited States, it is not (PUS/VA 100: 28 - 42 vs 40- 50),lower c value £ 0 subjectto regulatoryaction (Joseph Cavey, pers. comm.). (1.1 - 1.5 vs 1.6- 2.9),more elevated vulva, and six P. fallax was originallydistinguished from P.convalla- toeight lateral lines compared to four to six lines. The riae byitsshorter body length, narrower, more numerous excretorypore in P.arlingtoni n.sp. is often below the tailannules, and lower male to female ratio (Seinhorst, pharyngeal-intestinaljunction (p-ij), and is above the 1968). P. fallax was synonymisedby Frederickand T arjan p-ij in P.crenatus.P .arlingtoni n.sp. lacks a vulval (1989) with P. cerealis Haque,1965 because of thebelief, membrane,has a longerstylet (16 - 17.5 vs 15 - 16 ¹m) sharedby Loof(1978), that the  attenedcondition of the anda shorterpharyngeal overlap (6 - 31 vs 118 - 130 ¹m) typespecimens accounted for anartiŽ cially much lower compared to P. roseus. P.arlingtoni n.sp. differs from aratio.However, the original description of P. fallax also P. teres vs bymoreposterior vulva (V =81-86 69 - 78), distinguishedit from P. cerealis bya longerbody length fewer tailannules (19 - 25 vs 24-30),shorter pharyngeal (0.42 - 0.56 vs 0.39- 0.43mm), moreposterior excretory overlap(6 - 31 vs 58 ¹m), longerPUS/ VA 100 (28 - 42 £ pore(between nerve ring and pharyngeal-intestin alvalve vs 18)and six to eight lateral lines rather than only six. vs atlevel of medianbulb in P. cerealis),alongertail, and When P.arlingtoni n.sp.is compared to both P. conva- moreobscure,  atterlip annules (Seinhorst, 1968). llariae and P. fallax, ithasa lowerb value(4.1 - 5.3 vs 6.0 Recently,both P. fallax and P.convallariae were de- -9.0and5.2 - 6.7,respectively), and more posterior vulva tectedin a shipmentof C. majalis from France,through (V = 81 - 86 vs 78- 81and 77 - 81,respectively), lacks theNetherlands and destined for thestate of Ohio,USA. malesand has two to fourmore lateral lines. P.arlingtoni Mountedspecimens of originalparatypes of P. fallax were n.sp.also has more tail annules (19 - 25 vs 16 - 19) than comparedwith P.convallariae from apreviousintercep- P.convallariae .Thenewspecies has a longerstylet (16.0 - tion.Intermediate morphological forms were alsonoticed 17.5 vs 15.0 - 15.5 ¹m)moreoffset lip region, and coarser P.convallariae P. fallax tailannulation than P. fallax.Differencesfrom P. fallax betweenthe typical and . Pho- alsoapply to P.pseudofallax (Café-Filho & Huang,1989). tographsand supplemental measurements of representa- P.arlingtoni n.sp.differs from P.penetrans by having a tivemembers of eachspecies are provided and compared crenatetail in all specimens, lack of observedmales, lower withprevious descriptions. bvalue(4.1 - 5.3 vs 5.3- 7.9),shorter pharyngeal overlap The P. fallax populationfrom Francehad typical spe- (6 - 31 vs 32 - 65 ¹m), andlonger PUS/ VW(1.5- 2.5 vs cies-diagnosticcharacters of crenate tail terminus in all 1.0 - 1.5). membersof the population (compared to P.penetrans ), oblique,central lateral Ž eldstriations, body length (0.48 - 50.5 vs 0.50- 0.56mm inoriginaldescription vs 0.58 - Observations onintercepted Pratylenchus fallax 0.61 mm for P.convallariae ),roundedto anchor-shaped and P.convallariae styletknobs compared to more tulip-shaped knobs in P.convallariae ,tailannule numbers (19 - 26 vs 16 - 19 P. fallax iscommonin sandyor sandy-peatsoils (Sein- in P.convallariae ),andrare malesto distinguish it from horst,1977) around grass and ornamentals of many Eu- P.convallariae (Table2, Fig. 4) or P.penetrans where ropeancountries (W ebb,1990) as well as the Canadian malesare more numerous. Stylet lengths in this popula-

Vol.3(6), 2001 613 Z.A. Handoo et al.

Table 2. Morphometricsof Pratylenchusfallax and P.convallariae (afterSeinhorst, 1959 1, 19682, 19773; Loof, 19914. measure ¤ D derivedfrom original description). V aluesare in ¹m, andthose followed by ( n 10)were derived by the authors, and are presented D asrange followed by mean standarddeviation. § Measure P. fallax Seinhorst,1968 P.convallariae Seinhorst,1959 L 420 - 560 580 - 610 a 24 - 33 23 - 27 b 5.2 - 6.7 6.0 - 9.0 c 18 - 24 17 - 28 V 77 - 81 78 - 81 Styletlength 15 - 15.5 16 - 17 Tailannule number 16 - 26 16 - 19 Lateralline number 4,centre often oblique 4 Post-uterinesac/ Vulvalwidth 1.0 - 1.6 1.4 - 2.0 Post-uterinesac/ Vulva-anus 100 25 - 33 21 - 25 £ Pharyngealoverlap 20 - 44 (31)4 31 - 55 (41)4 Male:Femaleratio 1 : 5 up to 1 : 1 Spermathecashape round/ovalwhen empty round Lip number 3 3, offset Excretorypore position at/posteriorto nerve ring at/posteriorto nerve ring Phasmid-tailterminus 9-13annules 3 6* -8annules(n 10) D Pharynx 92 - 115 102:6 7:7 (n 10) 102 - 122 111 7:2 (n 10) § D § D Pharyngealoverlap 25 - 35 30:2 3:7 (n 10) 30 - 40 34:1 3:1 (n 10) § D § D b 4.3 - 5.2 4:8 0:4 (n 10) 4.0 - 4.8 4:4 0:2 (n 10) 0 § D § D c 1.7 - 2.2 2:0 0:2 (n 10) 2.0 - 2.4 2:2 0:1 (n 10) 0 § D § D Post-uterinesac 16 - 25 20:6 3:4 (n 10) 16 - 26 19:9 2:6 (n 10) § D § D Vulva-anusdistance 72 - 90 78:8 6:1 (n 10) 70 - 88 76:8 6:5 (n 10) § D § D Vulvalwidth 16 - 20 17:9 1:3 (n 10) 14 - 20 17:1 1:6 (n 10) § D § D Vulva-spermatheca 28 - 42 32:6 4:9 (n 10) 23 - 50 34:2 7:3 (n 10) § D § D Vulva-spermatheca/Vulva-anus 100 31 - 58 42 10 (n 10) 33 - 59 44 10 (n 10) £ § D § D tion(15.5 - 17 ¹m)overlappedthe narrow range for both quarterof the32 -65 ¹m range for P.penetrans overlap P. fallax (15 - 15.5 ¹m) and P.convallariae (16 - 17 ¹m). (Loof,1991). The number of tailannules from thetermi- The P. fallax populationalso had male tails with a slight nusto the phasmid was anotherdifferentiating character concavityon theposterior third of thebursa (Fig. 4I) asin between P. fallax (9- 13annules)and P.convallariae (less theoriginal drawing (Seinhorst, 1968). This P. fallax male thannine annules) based on literature descriptions and bursalproŽ le with the concavity after the papillar phas- observationsin specimens from thesetwo populations. midis different from theuniformly convex bursal pro- Exceptionalindividuals had P. fallax-liketail annulation, Žledescriptions (Seinhorst, 1959) and observations from with P.convallariae -likestylet knob-shape and length or P.convallariae (Fig.4N). Measurementranges (T able2), bifurcatedtail termini with more tail annules. One of supplementingthose in the literature, include c 0 and b0 theseindividuals had identical 28S rDNA D3sequences values,post-uterine sac length, vulva-anus distance, vulva tothose of completely true-to-type P.convallariae and width,vulva spermatheca and vulva-spermatheca relative P. fallax from separateshipments. Conversely, we mea- tovulva-anus distance. These measures are similar for sureda population(n = 10)with all the other characteris- P. fallax and P.convallariae ,butpharynx length or overlap tics of P.convallariae exceptfor smallerbody length(0.46 was slightlygreater in P.convallariae than P. fallax. The -0.58mm) thatbridges the originally non-overlapping pharyngealoverlaps occurred within the range of those publishedlength ranges for P. fallax (0.42- 0.56mm) alreadypublished (Loof, 1991). However, even the high- (Seinhorst,1959) and P.convallariae (0.58- 0.51mm) estvalue of the 30 - 40 ¹moverlap(average = 34 ¹m) (Seinhorst,1968). A voucherspecimen (UCDNC 3279) measuredhere for P.convallariae fellwithin the lower of this P. crenatus conformedto the species description.

614 Nematology Pratylenchusarlingtoni n. sp.

Fig. 4. Comparisonof Pratylenchusfallax and P.convallariae (lateralview), DIC A,B: P. fallax head; C: P. fallax lateralŽ eld,midbody; D: P. fallax femaletail; E: P. fallax maletail; F ,G: P.convallariae femaleheads; H: P.convallariae lateralŽ eld,mid-body; I, J: P.convallariae femaletails.

Molecularcharacterisation thesequence or 73inthe alignment, both within the vari- ableregion of thesequence. The third change, a Ginser- Shownin Fig. 5 arealigned sequences of theD3 region tionbetween G andT atposition251 in the sequence or oftheLS 28SrDNA for P.arlingtoni ,designatedas ‘ arl’, 256in thealignment, occurred in a highlyconserved re- for P.convallariae as‘ con’, andfor P. fallax as ‘fal’. gionof themolecule. The P.arlingtoni sequencediffered Alsoshown is the sequence for P.penetrans (Al-Banna by9/305bp (3%difference) from P.penetrans which in- et al.,1997)from theGenBank database that most nearly cludedthe previous changes, plus changes in common resembledour new sequences. with P. fallax and P.convallariae .Theseshared differ- The28S rDNA sequencesof typical specimens of encesamong the three species relative to P.penetrans in- P. convallariae and P. fallax were identical. P. arling- cludeda Csubstitutionfrom Gatposition 39 inthe align- toni n.sp.differed from thatsequence by threebase pairs ment,C substitutionfrom Aatposition 69, G from Tat (bp)/305(1% difference), which included a Gsubstitu- position71, T from Aatposition 82, G from Aatpo- tionfrom Aatposition 55 in the sequence or 56 in the sition84, C from Tatposition 139, G from Aatposi- alignment,and a Tsubstitutionfrom Catposition 71 in tion148, and A from Gatposition 231. The sequence

Vol.3(6), 2001 615 Z.A. Handoo et al.

Fig. 5. Sequencealignments of LS28SrDNAwith Clustal W for Pratylenchusarlingtoni n. sp., P.convallariae,P .fallax and P.penetrans. pen:Closestsequence in Genbank to the others, P.penetrans ,accessionnumber U47546 (Al-Banna et al., 1997); arl: P.arlingtoni ; con: P.convallariae ; fal: P. fallax;–:Gap.Nucleotides differing from the others are underlined and in bold. of P.fallax/P.convallariae differedfrom P.penetrans by ever, P.arlingtoni n.sp.is theŽ rst nematodewith a con- 12/305bp (4%).The sequence for P.crenatus, Accession sistentsix to eight,just as four to sixhave been found in a numberU47549 (not shown in T able5) (Al-Banna et al., few otherlesion nematode species. 1997)was fairlydistant from P.arlingtoni n.sp., differ- Pharyngealcharacters of P.arlingtoni n.sp. are im- ingby 49/305bp (16%),where all but six major changes portantfor itsidentiŽ cation. The pharyngeal overlap was occurredin a variableregion between alignment positions adiagnosticallyand phylogenetically reliable character 60 and 175. inwell-preservedspecimens in recentmorphological and molecularstudies with multiple Pratylenchus species (Loof,1991; Al-Banna et al.,1997;Duncan et al., 1999). Discussion Despitethe morphological similarity of P.arlingtoni n. sp. to P. crenatus populationsdescribed from Europe Theunusual character of sixor eightlateral lines was (Loof,1960, 1991; Urek, 1999) and South America (T or- reported(Roman & Hirschmann,1969) in a smallpro- res &Chaves,1999), there is substantial 28S rDNA se- portionof the populations of P.brachyurus (Godfrey, quencedifference between P.arlingtoni n.sp. and the 1929)Filipjev & SchuurmansStekhoven, 1941, P. coffeae P. crenatus populationfrom Oregon(Al Banna et al., (Zimmerman,1898) Filipjev & SchuurmansStekhoven, 1997)and a populationfrom Ohio(unpubl.). Just as Al- 1941, P.penetrans (Cobb,1917) Filipjev & Schuurmans Banna et al.(1997)proposed the genus Pratylenchus to Stekhoven,1941, P.scribneri Steiner,1943, P. vulnus bepolyphyletic, crenate-tailed nematodes with P. crena- Allen& Jensen,1951 and P. zeae Graham,1951. How- tus-likemorphology might be polyphyleticas well.

616 Nematology Pratylenchusarlingtoni n. sp.

Although P.arlingtoni n. sp. and P. crenatus are mor- Acknowledgements phologicallysimilar, they are molecularly distant. How- ever,the identical DNA sequenceshere for P. fallax and Theauthors thank Donna Ellington, Roberta Henegar P.convallariae couldlend support to aproposalthat P. fal- andSharon Ochs for technicalassistance, and Joseph lax beconsidereda subspeciesor synonymof P. conval- Caveyand Michael Firko, Animal and Plant Health In- lariae dueto morphological similarity. It would be prema- spectionService, USDA, Riverdale,MD, USA, for reg- tureto synonymise P. fallax as P.cerealis, assuggested by ulatoryinformation. The authors wish to thank Gerrit Frederickand T arjan(1989), when both species might be Karssen,Plant Protection Service, W ageningen,The Neth- bettersynonymised with P.convallariae, dueto overlap- erlands,for sharinginformation on P. fallax and P. conva- pingmorphological and molecular characters and prior- llariae.Theauthors thank Pierre Baujard,Gerrit Karssen ityof description.Breeding studies might be helpful since andMichel Luc for manuscriptreview. Mention of trade themorphologically intermediate specimens in thisreport namesor commercialproducts in this publication is solely mayhave been sterile hybrids of two species, or simply for thepurpose of providingspeciŽ c informationand does naturalvariants within a singlebiological species. notimply recommendation or endorsementby theUnited P. fallax was originallydescribed as being morpholog- StatesDepartment of Agriculture. icallysimilar to the highly variable P.penetrans (Sein- horst,1968), with fewer males,a generallylonger pharyn- References gealoverlap (Loof, 1991) andoccasional populations with crenatetails (T arté& Mai,1976) . P. fallax was alsodif- ferent from P.penetrans inlackingfertile hybrid progeny AL-BANNA, L., WILL IAMSON, V. & GARDNER,S.L.(1997). Phylogeneticanalysis of nematodes of the genus Praty- et al et al (Perry .,1980)and on isozyme gels (Ibrahim ., lenchus usingnuclear 26S rDNA. MolecularPhylogenetics 1995)or with restriction-enzyme-di gestedfragments of andEvolution 7, 94-102. therDNA ITS region(W aeyenberge et al.,2000).Here, it BALDWIN, J.G., FRISSE, L.M., VIDA, J.T., EDDLEMAN, appearsthat P. fallax mayshare at least as close a biologi- C.D. & THOMAS,W.K.(1997).An evolutionary framework calrelationship with P.convallariae as with P.penetrans. forthe study of developmental evolution in a setof nematodes Both P.convallariae and P. fallax haverelatively narrow, related to Caenorhabditiselegans. Molecular Phylogenetics overlappingmorphometric ranges compared to other le- andEvolution 8, 249-259. sionnematodes based on keysand information presented CAFÉ-FILHO, A.C. & HUANG,C.S.(1989).Description of Pratylenchuspseudofallax n.sp.with a keyto species of the here,and both are found with similar soil types, hosts and genus Pratylenchus Filipjev,1936 (Nematoda: Pratylenchi- overlappinggeographic regions. However, careful com- dae). Revuede Né matologie 12, 7-15. parativepathogenicity testing has been limited for P. con- CHOU, Q., RUSSELL, M., BIRCH, D.E., RAYMOND, J. & vallariae and P. fallax (Webb,1990). BLOCH,W.(1992). Prevention of pre-PCR mis-priming and Becauseof themorphological and molecular relation- primerdimerization improves low-copy-number ampliŽ ca- ship of P.arlingtoni n. sp., P. fallax and P.convallar- tions. NucleicAcids Research 20,1717-1723. iae,andtheir limited distribution or absencein theUnited CORBETT,D.C.M.(1970).Cereal Pratylenchus spp.in England andWales and their recognition. PlantP athology 19, 6-10. States,there is aneedfor furthersurvey of lesionnema- CORBETT,D.C.M.(1972).The effect of Pratylenchusfallax on todes,particularly in theUnited States, Canada and Eura- wheat,barley and sugar beet roots. Nematologica 18, 303- sia.It is difŽ cult to believethat P. fallax isrestricted to the 308. southernprovinces of Canadabut not the northern states DUNCAN, L.W., INSERRA, R.N., THOMAS, W.K., DUNN, D., oftheUnited States. However, if true, there may be apar- MUSTIKA, I., FRISSE, L.M., MENDES, M.L., MORRIS, allel in P. fallax beingcommon in Great Britain (Corbett, K. & KAPLAN,D.T. (1999).Genetic and morphological 1970),while both P.convallariae and P. fallax are found relationshipsamong isolates of Pratylenchuscoffeae and atlower latitudes in Europe (Seinhorst, 1959, 1977). The closelyrelated species. Nematropica 29, 61-80. ELLIS, R.E., SULSTON, J.E. & COULSON,A.R.(1986).The inuence of temperatureon morphological variability in rDNA of C. elegans:sequenceand structure. NucleicAcids thesespecies as currentlycharacterised might be consid- Research 14,2345-2364. ered.Further tests of European,Canadian and U.S. popu- FILIPJEV,I.N.(1936).On the classiŽ cation of theT ylenchinae. lationsof theserelated nematodes are needed to evaluate Proceedingsof the Helminthological Society of W ashington currentregulatory restrictions on P. fallax. 3, 80-82.

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FREDERICK, J.J. & TARJAN,A.C.(1989).A compendiumof SEINHORST,J.W.(1959). T wonew species of Pratylenchus . the genus Pratylenchus Filipjev,1936 (Nemata: Pratylenchi- Nematologica 4, 83-86. dae). Revuede Né matologie 12,243-256. SEINHORST,J.W.(1968). Three new Pratylenchus specieswith GOLDEN,A.M.(1990).Preparation and mounting nematodes adiscussionof thestructure of the cephalic framework and of formicroscopic observation. In: Zuckerman, B.M., Mai, W .F. thespermatheca in this genus. Nematologica 14,497-510. &Krusberg,L.R. (Eds). Plantnematology laboratory man- SEINHORST,J.W.(1977). Pratylenchusfallax .CIH descriptions ual. RevisedEdition. Amherst, MA, USA, Universityof ofplant parasitic nematodes, St Albans,UK, Commonwealth MassachusettsAgricultural Experimental Station, pp. 200- AgriculturalBureaux, Set 7, No. 98, pp. 1-2. 201. STIERNAGLE,T. (1999).Maintenance of C. elegans. In: Hope, GOLDEN, A.M., LOPEZ, CH.R. & VILCHEZ,R.H.(1992).De- I.A. (Ed). C. elegans, apracticalapproach .New York,NY , scriptionof Pratylenchusgutierrezi n.sp.(Nematoda: Praty- USA, OxfordUniversity Press, pp. 51-67. lenchidae)from coffee in Costa Rica. Journalof Nematology TARTÉ, R. & MAI,W.F. (1976).Morphological variation in 24,298-304. Pratylenchuspenetrans . Journalof Nematology 8, 185-195. HAQUE,M.M.(1965).T wonew species of plant nematodes. THOMPSON, J.D., HIGGINS, D.G. & GIBSON,T.J.(1994). DokladyTimiryazevskoi Selskokhozyaistrennoi Akademii CLUSTALW:improving the sensitivity of progressive 113,209-219. multiplesequence alignment through sequence weighting, position-speciŽc gappenalties and weight matrix choice. Nu- IBRAHIM, S.K., PERRY, R.N. & WEBB,R.M.(1995).Use cleicAcids Research ofisoenzyme and protein phenotypes. Annalsof Applied 22,4673-4680. TORRES, M.S. & CHAVES,E.J.(1999).Description of Biology 126,317-327. somePratylenchidae (Nemata) from Argentina. Nematologia KHAN, E. & SINGH,D.B.(1974).Five new species of Praty- Mediterranea 27,281-289. lenchus (Nematoda:Pratylenchidae) from India. IndianJour- UREK,G.(1999).The effect of host plants on thebiopotential nalof Nematol ogy4, 199-211. andthe morphometric characteristics of Pratylenchuscrena- LOOF,P.A.A.(1960).T axonomicstudies on the genus Praty- tus Loof, 1960. ActaPhytopathologica et Entomologica Hun- lenchus (Nematoda). Tijdschriftvoor Plantenziekten 66, 29- garica 34,253-262. 90. WAEYENBERGE, L., RYSS, A., MOENS, M., PINOCHET, J. LOOF,P.A.A.(1978).The genus Pratylenchus Filipjev,1936 & VRAIN,T.C.(2000).Molecular characterisation of 18 (Nematoda:Pratylenchidae): a reviewof its anatomy, mor- Pratylenchus speciesusing rDNA restrictionfragment length phology,distribution, systematics and identiŽ cation. Vaxtsky- polymorphism. Nematology 2, 135-142. ddsrapporter 5, 50 pp. WEBB,R.M.(1990).Effects of the nematode Pratylenchus LOOF,P.A.A.(1991).The family Pratylenchidae Thorne, 1949. fallax onrootsof oilseed rape ( Brassicanapus var. oleifera). In:Nickle, W .R.(Ed.). Manualof AgriculturalNematology . Revuede Né matologie 13,115-117. New York,NY ,USA, MarcelDekker Inc., pp. 363-421. WILL IAMS, B.D., SCHRANK, B., HUYNH, C., SHOWNKEEN, MINAGAWA,N.(1982).Descriptions of Pratylenchusgibbicau- R. & WATERSTON,R.H.(1992).A geneticmapping system datus n. sp. and P.macrostylus Wu,1971 (T ylenchida:Praty- in Caenorhabiditiselegans basedon polymorphic sequence- lenchidae)from Kyushu. JapaneseJournal of AppliedEnto- taggedsites. Genetics 131,609-624. mologyand Zoology 17,418-423. YU, Q., POTTER, J.W. & GILBY,G.(1998).Plant parasitic ne- NORTON,D.C.(1984). Distributionof plant-parasitic nema- matodesassociated with turfgrass in golf courses in Southern todespecies in North America .Hyattsville,MD, USA, So- Ontario. CanadianJournal of PlantP athology 20,304-307. cietyof Nematologists, 199 pp. ZARINA, B. & MAQBOOL,M.A.(1998).Descriptions and PERRY, R.N., PLOWRIGHT, R.A. & WEBB,R.M.(1980). observationson two new and two known species of the genus Matingbetween Pratylenchuspenetrans and P. fallax in Pratylenchus Filipjev,1936 (Nematoda: Pratylenchidae) from sterileculture. Nematologica 26,125-129. Pakistan. PakistanJournal of Nematology 16, 13-24. ROMAN, J. & HIRSCHMANN,H.(1969).Morphology and ZEIDAN, A.B. & GERAERT, E. (1991). Pratylenchus from morphometricsof six species of Pratylenchus . Journal of Sudan,with the description of two new species (Nemata: Nematology 1, 363-386. Tylenchida). Revuede Nématologie 14,261-275.

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