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

Identification of Seed Gall of Agronomic and Regulatory Concern with PCR-RFLP of ITS11

:I T. O. POWERS, A. L. SZALANSKI, G. MULLIN s. HARRIS,5 T. BERTOZZI AND GRIESBACH

Abstract: A molecular analysis of eight described of seed gall , along with six undescribed isolates from different hosts, has revealed a strong association betWeen nucleotide sequence polymorphism and host staws. Each anguinid nematode associated with a unique host produced a unique PCR-RFLP pattern for the ITSl region. spedes that had been synonymized in the past, Anguina agl'ostis, A. funesta, and A. 'tJevelli (Afrina 'tJeveni), were readily discriminated. Two undescribed species from northern New South Wales and southeastern South , 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, the vector 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, avenacea,Poopogon monospeliensis,StiPa sp., Astrebla pectinata, and Holcus lanatus. Phylogenetic analysis of the ITSl region suggests that considerable anguinid genetic diversification has accompanied specialization on different host species. Key words: Aftina IlJt!Velii,Angzlina agrostis,, diagnostics, nematode identification, PCR-RFLP, regulatory nematolo~', seed gall nematode.

Anguina species, the seed gall nematodes, have re- nid identification. Often, only juveniles are found in markably rich and convoluted taxonomic histories seed galls, which further complicates identification. (Brzeski, 1981; Fortuner and Maggenti, 1987; Krall, Undoubtedly, host records of different anguinid spe- 1991; Siddiqi, 2000). The genus includes the first re- cies are compromised b\' misidentification. The large corded plant parasite, Anguina trictici (Steinbuch, 1799) range of reported hosts for most likel, Fiiipjev, 1936, and A.funesta Price, Fisher, & Kerr, 1979, includes records for se,eral Anguina species (KI-all. the vector of the notoriousl)' toxic bacterium, Rathay- 1991). This study was initiated because of a need to ibacter toxicus. There are 11 valid species of Anguina rapidly identify anguinid species of regulatory concern. according to the latest classification (Siddiqi, 2000). A polymerase chain reaction-restriction fragment Species numbers, however, \.ary considerably among length polymorphism (PCR-RFLP) approach using the classifications, commensurate with the recognition of ribosomal DNA first internal transcribed spacer region various genera or subgenera. For example, Afrina (ITS1) has been found useful for DNA comparisons v,('(,(,zlivan den Berg, 1985, described originally from among individual nematodes (Powers et al., 1997). This \\eeping lovegrass, Agrostis CUl.vula,in South Mrica, was study reports surprising host specificity among all iso- synonymized with Anguina agrostis (Steinbuch, 1799) lates examined and pro\ides a convenient guide for Filipjev, 1936, the bentgrass nematode, by Chizhov and molecular recognition of anguinid species. Subbotin (1990). Ebsal")' (1991) did not recognize this synonymy and placed this species in the genus Suban- MATERIALS AND METHOD! guina Paramonov, 1967. Recently, Siddiqi (2000) trans- ferred the species to Anguina. These taxonomic actions Nematodesamples: The putative origin, host, and pro- ,..:-, not inconsequential when regulatory issues arise. vider for the anguinid isolates are presented in Table I, The synonymy of Afrina wevelliand Anguina agrostis im- National and international seed trade creates ambiguit\' posed quarantine status on seed gall nematodes found in determination of point-of-seed origin in some cases, on weeping lovegrass because of earlier synonymies of For example, one isolate of Anguina weveUiarrived ill A. agrostisand A. funesta (Siddiqi, 1985). our laboratory from USDA-APHIS offices in Oregoll Underlying the taxonomic confusion is a conserved after seed from South Africa was imported into Texas and nondescript morpholo~' that complicates angui- and subsequently shipped to Oregon for re-export. Se\"- eral nematology laboratories maintain vials of wheat seed galls for teaching purposes, although the last re- Received for p'lblication 16 March 2()(.l1 I Journal Series No. 13423, Agricullliral Research Di,ision, Univel"Sity of )\;e- ported collection of A, tntici in the United States was in braska-Lincoln. 1975 in Virginia (Carta, pers. comm.). Species determi- :I Associate Professor, Department of Plant Patholog} , University of Nebr...ka- nations were made by morphological examination of Lincoln. 'Assistant Professor, Department of Entomolog}', l'niversil)' of Arkansas, nematodes extracted from plant galls, host association. Favetteville, AR. :, Graduate Research Assistant aud "Research Technologist, Deparllnent of and geographic location. Additional biochemical and Plant Pathology, University of Nebraska-Lincoln. life-cycle irformation of Ausu"alian isolates is available ,; Evolutionary Biology Unit, 5<,uth Australian Museum, North Ten ace, from T. $ertozzi. Photographic vouchers and dried welaide 500(), Australia. 'Survey Plant Pathologist, Oregon Deparllnent of Agricullllf", Salem, OR galls of oi~her anguinid species are deposited in the E-mail: tpow"",@unlnotes,Ultl.edu University of Nebraska Department of Plant Patholo~' The authol"S thank K. Ball, M. Mc(;!ure, and T. Todd for providing nema. lodes nematology collection maintained by T. Powers. T!lis paper was "dited by B. (:. Hy,nan. DNA analysis: Single ju\"eniles were processed for PCR 191 P.A. 192 journal of Nematology,Volume 33, No.4, December2001

TABlE Seed gall nematode samples used for this study. 1980) of sequence evolution. Seed gall nematode DNA sequenceswere aligned using rDNA sequences from Specie Location Ditylenchusphyllobius (Thorne, 1934) Filipjev, 1936, D. .'inguina loeveli Agrostiscurvula, weeping South Mrica dipsici (Kuhn, 1857) Filipjev, 1936, and D. destructor lovegrass Thorne, 1945. GenBank accession numbers for the Anguina agrostis" Agrostis tenuis,bentgrass Oregon, U.S.A. three Ditylenchusspecies are AF363110 to AF363112. Agrof'Yronsmithii, western A nguina Kansas,U.S.A. Maximum likelihood and unweighted parsimony analy- agropy,"onifloris( wheatgrass -\nguina funestad Lalium rigidum, annual Australia sis of the alignments were conducted using PAUP* ryegrass 4.0b2 (Swofford, 1999). Gaps were excluded for all Angul Lalium rigidum, annual Geranium, analyses.Bootstrap analysis was used to compare the Australia ryegrass relative reliability of clades (Felsenstein, 1985). Parsi- funes/ad Lalium rigidum, annual Lochiel, ryegrass Australia mony bootstrap analysis included 1,000 resamplings .-inguina gramini.! Festucaovina var. Oregon, U.S.A. using the Branch and Bound algorithm of PAUP*. duriuscula, hard fescue For maximum likelihood analysis, either the single ,-Inguina Microlaenastipodes Australia microlaenaed.-inguina most-parsimonious tree generated in our parsimony pacijicae' PDQ annua, annual California, search '\'as used as the starting tree or a bootstrap bluegrass U.S.A. analysis ,vas conducted with 100 resamplings using .-inguina tntidf Triticum aestivum, wheat Baja, Mexico the Heuristic algorithm of PAUP*. Rateswere assumed .-inguina tntid' Triticum aestivum, wheat Baja, Mexico to follow a gamma distribution, \\ith shape parameter .-inguina tntici Triticum aestivum, wheat Virginia, U.S.A. 1975 estimated via maximum likelihood based on the -inguinasr. Dactylis glomerata, Oregon, U.S.A. general-time-reversible model (GTR) (Yang, 1994). A orchard grass total of 235 distinct data patterns were used for this .-\nguina sp. Agrostis avenacea,annual Australia blown grass model. inguina sp Polypogonmonospeliensis, Australia annual beardgrass .-inguinasr. StiPasr. Australia .-inguinasr. Astreblapectinata Australia .-inguinasr. Holcus lanatus,velvetgrass Australia DNA sequencing of the ITSI PCR-amplified product Collected by: .K. Ball, bJ. Griesbach, lSka Seed, d T. Bertozzi, .M. revealed a size range from 547 to 553 bp among eachof ~[caure, fT. Todd. the seed gall nematodes, with the exception of the an- guinid speciesfrom Astrebla,which produced a 575-bp b~' placing them in a 15-j-l1drop of distilled water on a product. No nucleotide differences were obser\'ed glass cover slip and manually disrupting them (Powers among the intraspecific comparisons. Unique amplifi- and Harris, 1993). Two PCR primers, rDNA2 (Vrain et cation products were obtained from every anguinid iso- al., 1992) and rDNA1.58S (Cherl"y et al., 1997) were late examined in this study that was extracted from a used to amplify a 3' portion of the 18S gene, the entire different host species (Tables 2,3). The two most simi- ITS 1 region, and a 5' section of the 5.8S gene. PCR lar amplification products came from un described spe- reactions were conducted per Szalanski et al. (1997). cies collected from annual beardgrass and annual .\mplified DNA for DNA sequencing was purified using blowngrass in Australia. The single substitution that Geneclean 11 (Bio 101, Inc., Vista, CA). The Iowa State separated the ITS1 amplified product of these two l'niversity DNA Sequencing and Synthesis Facility nematodes also affected an HhaI site, allowing their (Ames, IA) sequenced PCR products. Sequences were discrimination by this enzyme (Table 2). Table 2 obtained for both DNA strands, and consensus se- presentsa summary of digestion patterns from enzymes quences were derived from individual sequences in useful for anguinid identification. Table 3 presents a each direction using GAP (Genetics Computer Group, composite genotype representing the seven restriction Madison, WI). The GenBank Accession Numbers for patterns that permit convenient identification. Diges- the DNA sequences are AF363093 to AF363109. tion with the underlined enzymes in Table 3 provided Restriction sites were predicted from the DNA se- unambiguous identification of the nematodes included quence data using Web Cutter 2.0 (Heiman, 1997). Am- in this study. Restriction digestion of amplified DNA plified DNA was digested according to manufacturer's from nematodes extracted from a wheat gall collected (New England Biolabs, Beverly, MA) recommendations in 1974 resulted in restriction patterns identical to A. per Cherry et al. (1997) using the restriction enZ}"ffies tritici patterns from recently collected galls. Alu I, Bsr I, Eco RI, Hae 1lI, Hha I, Hinf I, and Taq I. The rDNA sequences of all of the seed gall nema- Fragments were separated byagarose (2.5% MetaPhor) todes were aligned using three Ditylenchusspp. as the gel electrophoresis following Cherry et al. (1997). outgroup taxa. The aligned data matrix including the The distance matrix feature of PAUP* 4.0b2 (Swof- outgroup taxa resulted in a total of 721 characters, in- ford, 1999) was used to calculate genetic distances ac- cluding gaps, and is available at nematology.unl.edu/ cording to the Kimura 2-parameter model (Kimura, ang-uina.htm.Of these characters, 269 (37%) were vari-

RESULTS-inguina Seed Gall Nematode Identification: Powerset at. 193

TABLE 2.. Seed gall nematode restriction fragment sizesand patterns.

able and 124 (17%) were informative for parsimony. tions in regulatory settings where misidentification can Bootstrap analysis of the aligned seed gall and Di- result in a significant economic impact. Several of the tylenchusspp. rDNA ITS1 sequences resulted in a con- samples provided for this study were actually nema- sensus tree (Length = 529, CI = 0.781), which delin- todes intercepted on shipments of seed. Anguina weuelli eated four clades (Fig. I). Regardless of whether the has been intercepted in the United States on imported .starting tree was the most parsimonious one or was ob- shipments of \\'eeping love grass seed, Agrostis curvula, tained \ia step-wise addition, the maximum likelihood on several occasions. The possibility that this nematode search found only one tree (Fig. 2). This maximum may be identical to A. funesta, as indicated by Krall likelihood tree (-Ln likelihood = 3441.96403, total (1991), restricts the movement of this seed. The ITS1 number of rearrangements tried = 2,920) was identical in topology to the parsimony tree, and to the maximum likelihood U"eederived by bootstrap analysis. 57 DISCUSSION Amplification and digestion of the ITSI region could distinguish each of the anguinid speciesexamined dur- ing this study. As a diagnostic t061, this process could eliminate much of the ambiguity involved in morpho- logical identification of juvenile specimens. Anguinid specimens are usually encountered as juveniles in galled plant tissue or residue following seed testing for germination (Griesbach et al., 1999). Importantly, this method could provide standardization for identifica-

TABLE 3. Seed gall nematode patterns for the restriction en- zyrnesAlu I Bsr I, EcoR I, Hae III, Hha I, Hinf I. and Taq I.

Sample

Anguina lvevelli A. agrostis A. aglvpyronifloris A. junesla A. graminis A. microlaenae AngtJina microlaenae A. pacijicae destructor A. tritici

A. sp. Dactylis -Ditylenchus phyllobius A. sp. Agrostis A. sp. Polypogon ---L- Ditytenchusdipsaci A. sp. StiPa FIG. I. Single most-parsimonious tree found using a branch and A. sp. Astrebia bound search using PAUP* of rDNA ITS I sequences with gapped A. sr. Holcus characters excluded. Bootstrap values for 1,000 branch and boltnd replicates are listed above branches supported at ~50%.

~I 194 journal of Nematology, Volume 33, No.4, Decl'mber2001

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

Anguina tritica Mexico Man, of the nematodes included in this study are seldom encountered in the field. Other than A. funesta I I Anguinatriticl Mexico2 and A. tritici, all of the anguinid species examined in L Anguinaagropyronilloris this study came from a restricted geographic locality. Currentlv, we are attempting to collect anguinid-in- rl Anguinasp Agrostis1 cluced galls that permit a more extensive comparison of l Anguinasp Polypogoni geographic and temporal variation.

lo-- Anguina graminis LITERA TURE CITED I Anguina funesta1 Bertozzi. T., and A. C. McKay. 1995. Incidence on Polypogon monl>- speliensis of Rathyaibarlertoxicus and Anguina sp., the organisms asso- Anguina funesta Geranium ciated \\ith floodplain staggers in south Australia. Australian Journal Anguina funesta Lochiel of Experimental Agriculture 35:567-569. Brzeski. \-1. 1981 The genera of (Nematoda, Tvlen-

-Anguina sp Holcus chida). Revue de :'-;ematologie 4:23-24. Cheri",. T., A. L. Szalanski, T. C. Todd, and T. O. Po\\'ers. 1997. The Anguina agrostis internal transcribed spacer region of (!\emata: Belono- laimidae\. Journal of :-iematology 29:21-29. Chizhov. V. N., and S. A. Subbotin, 1990. Phytoparasitic nematodes of the subfamily Anguininae (Nematoda: ). Morphology. trophic specialization, systematics. Zoologeschki Zhurnal 69:15-26. Anguinamicrolaenae Ebsar\. B. A. 1991. Catalog of the order Tylenchida (Nematoda). Research Branch, Agriculture Canada. Felsenstein, J. 1985. Confidence limits on phylogenies: An ap- Dilylenchus destructor proach using the bootstrap. Evolution ~9:783-791. Fortuner, R., and A. Maggenti. 1987. A reappraisal ofTylenchina ., Ditylenchus phyllobius (~emata). 4. The family Anguinidae Nicoll, 1935 (1926). Re\"tle de

-':- i,'. Kematologie 10:163-176. 005 substitutions/site Griesbach,J.,J.J. Chitambar, M.J. Hamerlynck, and E. O. Duarte F/{;. 2. Best maximum likelihood tree based on rDNA ITS I se- 1999. A comparative analysis of extraction methods for the recover\ quences. Branch lengths are proportional to the number of inferred of Anguina sp. from grass seed samples. Journal of !\ematolo~ 33: 635-640. changes. Heiman, M. 1997. Webcutter 2.0. http://www.ccsi.com/firstmarket/ firstmarket/ cutter / cut2.html. nucleotide sequence of A. wevelli, A. fimesta, and A. Kimura, M. 1980. A simple method for estimating evolutionary agrostis clearly demonstrates that these are all separate rates of base substitutions through comparative studv of nucleotide species. The phylogenetic analysis presented in this pa- sequences. Journal of Molecular Evolution 16: 111-120. Krall, E. L. 1991. vVheat and grass nematodes: Anguina, Suban- per suggests that A. wevelli is a member of a grouping gzlina, and related genera. Pp. 721-760 in W. R. Nickle. ed. Manual of separate from those species and possibly separate from Agricultllral Nematology. New York: Marcel Dekk~r. other nominal species in Anguina. Powers, T. 0., and T. S. Harris. 1993. A polymerase chain reaction method for identification of five major Meloidogynespecies. Journal of Shipments of seed of orchardgrass, Dactylis glomerata, Nematology 25:1-6. have been rejected based on the presence of a nema- Powers, T.O., T. C. Todd, A. M. Burnell, P. C. B. \furray, C. C. tode believed to be A. agrostis. Digestion of the ITS1 Fleming, A. L. Szalanski, B. A. Adams, and T. S. Harris. 1997. The amplification product with EcoR I, Hae III, or Hinf I Internal Transcribed Spacer Region as a Taxonomic Marker for Nematodes. Journal of Nematology 29:441-450. differentiates between the anguinid isolate from or- Siddiqi, M. R. 1985. Tylenchida: Parasites of plants and insects, I" chardgrass and A. agl'Ostis.The sensitivity of the PCR- ed. Farnham Royal, Slough: Commonwealth Institute of Parasitology. RFLP approach is illustrated by the discrimination of Siddiqi, M. R. 2000. Tylenchida: Parasites of plants and insects, 2nd the two tmdescribed anguinid species associated with ed. Oxon, U.K.: C-\BI Publishing, CAB International. Swofford, D. L. 1999. PAUP*. Phylogenetic analysis using parsi- "floodplain staggers" (Bertozzi and McKa~', 1995). A mony (*and other methods), verso 4. MA: Sunderlan.c\. single G..-:,Asubstitution in ITS1 is the onl~' nucleotide Szalanski, A. L., D. D. Sui, T. S. Harris, and T. O. Powers. 1997 difference obsef\'ed between these nematodes, vet the Identification of cyst nematodes of agronomic and regulatory con- creation of an Hha I site in the isolate found on annual 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. blowngrass, Agrostis avenacea,provided a means to dif- 1992. Intraspecific rD:-iArestriction fragment length polymorphism ferentiate it from the isolate found associated \\'ith an- in the Xiphinema alnencanum group. Fundamental and Applied Nema- nual beardgrass, Polypogon monospeliensis. tology 15:563-573. It is tempting, based on the phylogenetic analysis of \'ang, Z. 1994. Maximum likelihood phylogenetic estimation from D~A sequences with variable rates over sites: Approximate methods. these nematodes, to imagine that specialization on dif- journal of Molecular Evohltion 39:306-314. ~-Lrl,