Journal of Nematology 33(4):191-194.2001@ The Society of Nematologists200 I

Identification of Seed Gall Nematodes of Agronomic and RegulatoryConcern with PCR-RFLP of ITS11

:IT. O. POWERS, A. L. SZALANSKI,

P.

G. MULLIN s. HARRIS,5 T. BERTOZZI

A.

GRIESBACHAND

Abstract: A molecular analysis of eight described species of seed gall nematode, along with six undescribed isolates from differenthosts, has revealed a strong association betWeen nucleotide sequence polymorphism and host staws. Each anguinid nematodeassociated with a unique host produced a unique PCR-RFLP pattern for the ITSl region. Anguina spedes that had been synonymizedin the past, Anguina agl'ostis, A. funesta, and A. 'tJevelli (Afrina 'tJeveni), were readily discriminated. Two undescribed species fromnorthern New South Wales and southeastern South Australia, reported to be vectors of Rathyaibactl'1" toxicus in the disease called"floodplain staggers," "'ere differentiated by a single restrIction en~'llie. and both could be separated easily from A. funesta, thevector of R. toxicus in annuall,'egrass toxicity. Other species differentiated in this study include .1. agropyronifloris, A. graminis, A.miC1.olaenae, A. pacificae, and un described species from host species Dactylis glomerata, Agrostis avenacea, Poopogon monospeliensis, StiPasp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITSl region suggests that considerable anguinid geneticdiversification has accompanied specialization on different host species.

Key words: Aftina IlJt!Velii, Angzlina agrostis, Anguina tritici, diagnostics, nematode identification, PCR-RFLP, regulatory nematolo~',seed gall nematode.

nid identification. Often, only juveniles are found inseed galls, which further complicates identification.Undoubtedly, host records of different anguinid spe-cies are compromised b\' misidentification. The largerange of reported hosts for Anguina agrostis most likel,includes records for se,eral Anguina species (KI-all.1991). This study was initiated because of a need torapidly identify anguinid species of regulatory concern.A polymerase chain reaction-restriction fragment

length polymorphism (PCR-RFLP) approach using theribosomal DNA first internal transcribed spacer region(ITS1) has been found useful for DNA comparisonsamong individual nematodes (Powers et al., 1997). Thisstudy reports surprising host specificity among all iso-lates examined and pro\ides a convenient guide formolecular recognition of anguinid species.

MATERIALS AND METHOD!

Anguina species, the seed gall nematodes, have re-markably rich and convoluted taxonomic histories(Brzeski, 1981; Fortuner and Maggenti, 1987; Krall,1991; Siddiqi, 2000). The genus includes the first re-corded plant parasite, Anguina trictici (Steinbuch, 1799)Fiiipjev, 1936, and A.funesta Price, Fisher, & Kerr, 1979,the vector of the notoriousl)' toxic bacterium, Rathay-ibacter toxicus. There are 11 valid species of Anguinaaccording to the latest classification (Siddiqi, 2000).

Species numbers, however, \.ary considerably amongclassifications, commensurate with the recognition ofvarious genera or subgenera. For example, Afrinav,('(,(,zli van den Berg, 1985, described originally from\\eeping lovegrass, Agrostis CUl.vula, in South Mrica, wassynonymized with Anguina agrostis (Steinbuch, 1799)Filipjev, 1936, the bentgrass nematode, by Chizhov andSubbotin (1990). Ebsal")' (1991) did not recognize thissynonymy and placed this species in the genus Suban-guina Paramonov, 1967. Recently, Siddiqi (2000) trans-ferred the species to Anguina. These taxonomic actions,..:-, not inconsequential when regulatory issues arise.The synonymy of Afrina wevelli and Anguina agrostis im-posed quarantine status on seed gall nematodes foundon weeping lovegrass because of earlier synonymies ofA. agrostis and A. funesta (Siddiqi, 1985).

Underlying the taxonomic confusion is a conservedand nondescript morpholo~' that complicates angui-

Received for p'lblication 16 March 2()(.l1I Journal Series No. 13423, Agricullliral Research Di,ision, Univel"Sity of )\;e-

braska-Lincoln.:I Associate Professor, Department of Plant Patholog} , University of Nebr...ka-

Lincoln.'Assistant Professor, Department of Entomolog}', l'niversil)' of Arkansas,

Favetteville, AR.:, Graduate Research Assistant aud "Research Technologist, Deparllnent of

Plant Pathology, University of Nebraska-Lincoln.,; Evolutionary Biology Unit, 5<,uth Australian Museum, North Ten ace,

welaide 500(), Australia.'Survey Plant Pathologist, Oregon Deparllnent of Agricullllf", Salem, ORE-mail: tpow"",@unlnotes,Ultl.eduThe authol"S thank K. Ball, M. Mc(;!ure, and T. Todd for providing nema.

lodesT!lis paper was "dited by B. (:. Hy,nan.

Nematode samples: The putative origin, host, and pro-vider for the anguinid isolates are presented in Table I,National and international seed trade creates ambiguit\'in determination of point-of-seed origin in some cases,For example, one isolate of Anguina weveUi arrived illour laboratory from USDA-APHIS offices in Oregollafter seed from South Africa was imported into Texasand subsequently shipped to Oregon for re-export. Se\"-eral nematology laboratories maintain vials of wheatseed galls for teaching purposes, although the last re-ported collection of A, tntici in the United States was in1975 in Virginia (Carta, pers. comm.). Species determi-nations were made by morphological examination ofnematodes extracted from plant galls, host association.and geographic location. Additional biochemical andlife-cycle irformation of Ausu"alian isolates is availablefrom T. $ertozzi. Photographic vouchers and driedgalls of oi~her anguinid species are deposited in theUniversity of Nebraska Department of Plant Patholo~'nematology collection maintained by T. Powers.

DNA analysis: Single ju\"eniles were processed for PCR

191

192 journal of Nematology, Volume 33, No.4, December 2001

1980) of sequence evolution. Seed gall nematode DNAsequences were aligned using rDNA sequences fromDitylenchus phyllobius (Thorne, 1934) Filipjev, 1936, D.dipsici (Kuhn, 1857) Filipjev, 1936, and D. destructorThorne, 1945. GenBank accession numbers for thethree Ditylenchus species are AF363110 to AF363112.Maximum likelihood and unweighted parsimony analy-sis of the alignments were conducted using PAUP*4.0b2 (Swofford, 1999). Gaps were excluded for allanalyses. Bootstrap analysis was used to compare therelative reliability of clades (Felsenstein, 1985). Parsi-mony bootstrap analysis included 1,000 resamplingsusing the Branch and Bound algorithm of PAUP*.For maximum likelihood analysis, either the singlemost-parsimonious tree generated in our parsimonysearch '\'as used as the starting tree or a bootstrapanalysis ,vas conducted with 100 resamplings usingthe Heuristic algorithm of PAUP*. Rates were assumedto follow a gamma distribution, \\ith shape parameterestimated via maximum likelihood based on thegeneral-time-reversible model (GTR) (Yang, 1994). Atotal of 235 distinct data patterns were used for thismodel.

Seed gall nematode samples used for this study.TABlE

Specie Location

South Mrica.'inguina loeveli

Oregon, U.S.A.Kansas, U.S.A.

Anguina agrostis"A nguina

agropy,"onifloris(-\nguina funestad Australia

Geranium,Australia

Lochiel,Australia

Oregon, U.S.A.

Angul

-inguina

funes/ad

.-inguina gramini.!

Agrostis curvula, weepinglovegrass

Agrostis tenuis, bentgrassAgrof'Yron smithii, western

wheatgrassLalium rigidum, annual

ryegrassLalium rigidum, annual

ryegrassLalium rigidum, annual

ryegrassFestuca ovina var.

duriuscula, hard fescueMicrolaena stipodes Australia,-Inguina

microlaenaed.-inguina

pacijicae' California,U.S.A.

Baja, MexicoBaja, MexicoVirginia,

U.S.A. 1975Oregon, U.S.A.

PDQ annua, annual

bluegrassTriticum aestivum, wheatTriticum aestivum, wheatTriticum aestivum, wheat

-inguina sr.

Australia.-\nguina sp.

Australiainguina sp

RESULTS

.-inguina sr.

.-inguina sr.

.-inguina sr.

Dactylis glomerata,orchard grass

Agrostis avenacea, annualblown grass

Polypogon monospeliensis,annual beardgrass

StiPa sr.Astrebla pectinataHolcus lanatus, velvetgrass

AustraliaAustraliaAustralia

lSka Seed, d T. Bertozzi, .M.Collected by: .K. Ball, b J. Griesbach,

~[caure, fT. Todd.

DNA sequencing of the ITSI PCR-amplified productrevealed a size range from 547 to 553 bp among each ofthe seed gall nematodes, with the exception of the an-guinid species from Astrebla, which produced a 575-bpproduct. No nucleotide differences were obser\'edamong the intraspecific comparisons. Unique amplifi-cation products were obtained from every anguinid iso-late examined in this study that was extracted from adifferent host species (Tables 2,3). The two most simi-lar amplification products came from un described spe-cies collected from annual beardgrass and annualblowngrass in Australia. The single substitution thatseparated the ITS1 amplified product of these twonematodes also affected an HhaI site, allowing theirdiscrimination by this enzyme (Table 2). Table 2presents a summary of digestion patterns from enzymesuseful for anguinid identification. Table 3 presents acomposite genotype representing the seven restrictionpatterns that permit convenient identification. Diges-tion with the underlined enzymes in Table 3 providedunambiguous identification of the nematodes includedin this study. Restriction digestion of amplified DNAfrom nematodes extracted from a wheat gall collectedin 1974 resulted in restriction patterns identical to A.tritici patterns from recently collected galls.

The rDNA sequences of all of the seed gall nema-todes were aligned using three Ditylenchus spp. as theoutgroup taxa. The aligned data matrix including theoutgroup taxa resulted in a total of 721 characters, in-cluding gaps, and is available at nematology.unl.edu/ang-uina.htm. Of these characters, 269 (37%) were vari-

b~' placing them in a 15-j-l1 drop of distilled water on aglass cover slip and manually disrupting them (Powersand Harris, 1993). Two PCR primers, rDNA2 (Vrain etal., 1992) and rDNA1.58S (Cherl"y et al., 1997) wereused to amplify a 3' portion of the 18S gene, the entireITS 1 region, and a 5' section of the 5.8S gene. PCRreactions were conducted per Szalanski et al. (1997)..\mplified DNA for DNA sequencing was purified usingGeneclean 11 (Bio 101, Inc., Vista, CA). The Iowa Statel'niversity DNA Sequencing and Synthesis Facility(Ames, IA) sequenced PCR products. Sequences wereobtained for both DNA strands, and consensus se-quences were derived from individual sequences ineach direction using GAP (Genetics Computer Group,Madison, WI). The GenBank Accession Numbers forthe DNA sequences are AF363093 to AF363109.

Restriction sites were predicted from the DNA se-quence data using Web Cutter 2.0 (Heiman, 1997). Am-plified DNA was digested according to manufacturer's(New England Biolabs, Beverly, MA) recommendationsper Cherry et al. (1997) using the restriction enZ}"ffiesAlu I, Bsr I, Eco RI, Hae 1lI, Hha I, Hinf I, and Taq I.Fragments were separated byagarose (2.5% MetaPhor)gel electrophoresis following Cherry et al. (1997).

The distance matrix feature of PAUP* 4.0b2 (Swof-ford, 1999) was used to calculate genetic distances ac-cording to the Kimura 2-parameter model (Kimura,

.-inguina tntidf.-inguina tntid'.-inguina tntici

193Seed Gall Nematode Identification: Powers et at.

TABLE 2.. Seed gall nematode restriction fragment sizes and patterns.

able and 124 (17%) were informative for parsimony.Bootstrap analysis of the aligned seed gall and Di-tylenchus spp. rDNA ITS1 sequences resulted in a con-sensus tree (Length = 529, CI = 0.781), which delin-eated four clades (Fig. I). Regardless of whether the.starting tree was the most parsimonious one or was ob-tained \ia step-wise addition, the maximum likelihoodsearch found only one tree (Fig. 2). This maximumlikelihood tree (-Ln likelihood = 3441.96403, totalnumber of rearrangements tried = 2,920) was identicalin topology to the parsimony tree, and to the maximumlikelihood U"ee derived by bootstrap analysis.

tions in regulatory settings where misidentification canresult in a significant economic impact. Several of thesamples provided for this study were actually nema-todes intercepted on shipments of seed. Anguina weuellihas been intercepted in the United States on importedshipments of \\'eeping love grass seed, Agrostis curvula,on several occasions. The possibility that this nematodemay be identical to A. funesta, as indicated by Krall(1991), restricts the movement of this seed. The ITS1

I

57DISCUSSION

Amplification and digestion of the ITSI region coulddistinguish each of the anguinid species examined dur-ing this study. As a diagnostic t061, this process couldeliminate much of the ambiguity involved in morpho-logical identification of juvenile specimens. Anguinidspecimens are usually encountered as juveniles ingalled plant tissue or residue following seed testing forgermination (Griesbach et al., 1999). Importantly, thismethod could provide standardization for identifica-

~

TABLE 3.

zyrnesAlu I

Seed gall nematode patterns for the restriction en-Bsr I, EcoR I, Hae III, Hha I, Hinf I. and Taq I.

Sample

AngtJina microlaenae

Ditylenchus destructor

Anguina lvevelliA. agrostisA. aglvpyroniflorisA. juneslaA. graminisA. microlaenaeA. pacijicaeA. triticiA. sp. DactylisA. sp. AgrostisA. sp. PolypogonA. sp. StiPaA. sp. AstrebiaA. sr. Holcus

-Ditylenchus phyllobius

---L- Ditytenchus dipsaci

FIG. I. Single most-parsimonious tree found using a branch andbound search using PAUP* of rDNA ITS I sequences with gappedcharacters excluded. Bootstrap values for 1,000 branch and boltndreplicates are listed above branches supported at ~50%.

194 journal of Nematology, Volume 33, No.4, Decl'mber 2001

r-- Anguina sp Stipa ferent host species has served as an isolating mecha-nism in anguinid evolution. Greater resolution of spe'-cies relations and more conspecific populations arenecessary before theories of speciation can be tested.

Man, of the nematodes included in this study areseldom encountered in the field. Other than A. funestaand A. tritici, all of the anguinid species examined inthis study came from a restricted geographic locality.Currentlv, we are attempting to collect anguinid-in-cluced galls that permit a more extensive comparison ofgeographic and temporal variation.

r-'-;L-- Anguina wevell;

rl

Anguina sp Astrebla

Anguina tritica Mexico

I I Anguina triticl Mexico2

L Anguina agropyronilloris

rl Anguina sp Agrostis1

l Anguina sp Polypogoni

lo-- Anguina graminis

I Anguina funesta1

Anguina funesta Geranium

Anguina funesta Lochiel

~

-Anguina sp Holcus

Anguina agrostis

,

Anguina microlaenae

Dilylenchus destructor

Ditylenchus phyllobius.,

-L

-':- i,'. Ditylenchus dipsaci005 substitutions/site

F/{;. 2. Best maximum likelihood tree based on rDNA ITS I se-quences. Branch lengths are proportional to the number of inferred

changes.

LITERA TURE CITED

Bertozzi. T., and A. C. McKay. 1995. Incidence on Polypogon monl>-speliensis of Rathyaibarlertoxicus and Anguina sp., the organisms asso-ciated \\ith floodplain staggers in south Australia. Australian Journalof Experimental Agriculture 35:567-569.

Brzeski. \-1. 1981 The genera of Anguinidae (Nematoda, Tvlen-chida). Revue de :'-;ematologie 4:23-24.

Cheri",. T., A. L. Szalanski, T. C. Todd, and T. O. Po\\'ers. 1997. Theinternal transcribed spacer region of Belonolaimus (!\emata: Belono-laimidae\. Journal of :-iematology 29:21-29.

Chizhov. V. N., and S. A. Subbotin, 1990. Phytoparasitic nematodesof the subfamily Anguininae (Nematoda: Tylenchida). Morphology.

trophic specialization, systematics. Zoologeschki Zhurnal 69:15-26.Ebsar\. B. A. 1991. Catalog of the order Tylenchida (Nematoda).

Research Branch, Agriculture Canada.Felsenstein, J. 1985. Confidence limits on phylogenies: An ap-

proach using the bootstrap. Evolution ~9:783-791.Fortuner, R., and A. Maggenti. 1987. A reappraisal ofTylenchina

(~emata). 4. The family Anguinidae Nicoll, 1935 (1926). Re\"tle deKematologie 10:163-176.

Griesbach,J.,J.J. Chitambar, M.J. Hamerlynck, and E. O. Duarte1999. A comparative analysis of extraction methods for the recover\of Anguina sp. from grass seed samples. Journal of !\ematolo~ 33:635-640.

Heiman, M. 1997. Webcutter 2.0. http://www.ccsi.com/firstmarket/firstmarket/ cutter / cut2.html.

Kimura, M. 1980. A simple method for estimating evolutionaryrates of base substitutions through comparative studv of nucleotidesequences. Journal of Molecular Evolution 16: 111-120.

Krall, E. L. 1991. vVheat and grass nematodes: Anguina, Suban-gzlina, and related genera. Pp. 721-760 in W. R. Nickle. ed. Manual ofAgricultllral Nematology. New York: Marcel Dekk~r.

Powers, T. 0., and T. S. Harris. 1993. A polymerase chain reactionmethod for identification of five major Meloidogyne species. Journal ofNematology 25:1-6.

Powers, T.O., T. C. Todd, A. M. Burnell, P. C. B. \furray, C. C.Fleming, A. L. Szalanski, B. A. Adams, and T. S. Harris. 1997. TheInternal Transcribed Spacer Region as a Taxonomic Marker forNematodes. Journal of Nematology 29:441-450.

Siddiqi, M. R. 1985. Tylenchida: Parasites of plants and insects, I"ed. Farnham Royal, Slough: Commonwealth Institute of Parasitology.

Siddiqi, M. R. 2000. Tylenchida: Parasites of plants and insects, 2nded. Oxon, U.K.: C-\BI Publishing, CAB International.

Swofford, D. L. 1999. PAUP*. Phylogenetic analysis using parsi-mony (*and other methods), verso 4. MA: Sunderlan.c\.

Szalanski, A. L., D. D. Sui, T. S. Harris, and T. O. Powers. 1997Identification of cyst nematodes of agronomic and regulatory con-cern by PCR-RFLP of ITS I. Journal of Nematology 29:25.'i-267.

\rain, T. C., D. A. Wakarchuk, A. C. Levesque, and R.I. Hamilton.1992. Intraspecific rD:-iArestriction fragment length polymorphismin the Xiphinema alnencanum group. Fundamental and Applied Nema-tology 15:563-573.

\'ang, Z. 1994. Maximum likelihood phylogenetic estimation fromD~A sequences with variable rates over sites: Approximate methods.journal of Molecular Evohltion 39:306-314.

nucleotide sequence of A. wevelli, A. fimesta, and A.agrostis clearly demonstrates that these are all separatespecies. The phylogenetic analysis presented in this pa-per suggests that A. wevelli is a member of a groupingseparate from those species and possibly separate fromother nominal species in Anguina.

Shipments of seed of orchardgrass, Dactylis glomerata,have been rejected based on the presence of a nema-tode believed to be A. agrostis. Digestion of the ITS1amplification product with EcoR I, Hae III, or Hinf Idifferentiates between the anguinid isolate from or-chardgrass and A. agl'Ostis. The sensitivity of the PCR-RFLP approach is illustrated by the discrimination ofthe two tmdescribed anguinid species associated with

"floodplain staggers" (Bertozzi and McKa~', 1995). Asingle G..-:,A substitution in ITS1 is the onl~' nucleotidedifference obsef\'ed between these nematodes, vet thecreation of an Hha I site in the isolate found on annual

blowngrass, Agrostis avenacea, provided a means to dif-ferentiate it from the isolate found associated \\'ith an-nual beardgrass, Polypogon monospeliensis.

It is tempting, based on the phylogenetic analysis ofthese nematodes, to imagine that specialization on dif-