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, Nematology Laboratory, 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 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 identi- 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 lterpaper in Baermann Thepurpose of thisstudy was tomorphologicallyand funnels.Specimens were thenhandpicked and xedin molecularlydescribe the new nematode and compare it 3%formalin. Some specimens were studiedin 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 speci ed. Light micro- scopicimages of live and 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 MORPHOLOGICAL CHARACTERISATION livenematodes were takenwith an imaging system em- P.arlingtoni n.sp. was foundin turf ( Poapratensis ployingImage-Pro Plusver 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 products were 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 puried 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 ora 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 oconrm theau- thenticityof the sequences obtained, PCR ampli cation 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 AMPLIFICATION AND SEQUENCING 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 amplied 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 followingmodi cations. Manufacturer-supplied Display- and P. fallax havebeen deposited in the GenBank data- TAQ buffer(PGC Scientic, 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 nwaxwas 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 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 sufcient shownin the alignment provided here (Fig. 5). Positions amountof PCR product. Prior tosequencing, the DNA are numberedwhere 1 correspondsto number 3324 of was puried usingthe Qiaquick PCR puri cation 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 product and 3.2 pmolD3A orD3B primer. T o tionof nucleotide differences among the four taxa were generatecloned PCR products for 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 eldbegin- ningbehind the level of thestylet as fournarrow crenate MEASUREMENTS lines,widening to veby the level of the median bulb, andsix to eight by the anterior