Journal of Nematology 29 (4) :441-450. I997. © The Society of Nematologists 1997.

The rDNA Internal Transcribed Spacer Region as a Taxonomic Marker for 1

T. O. POWERS,2 T. C. TODD, ~ A. M. BURNELL, 4 P. C. B. MURRAY, 4 C. C. FLEMING, 5 A. L. SZALANSKI,6 B. A. ADAMS,6 AND T. S. HARRIS6

Abstract: The ITS region from a wide taxonomic range of nematodes, including secernentean and adenophorean taxa, and free-living, entomopathogenic, and plant-parasitic species, was evaluated as a taxonomic marker. Size of the amplified product aided in the initial determination of group member- ship, and also suggested groups that may require taxonomic reevaluation. Congeneric species often displayed identically sized ITS regions, but genera such as Pratylenchus and Tylenchorhynchushad species with large differences in size. ITS heterogeneity in individuals and populations was identified in several taxa. PCR-RFLP of ITS1 is advocated as a method of taxonomic analysis in genera such as Helicotylenchus that contain numerous species with few diagnostic morphological characteristics. Key words: diagnosis, genetics, internal transcribed spacer region, ITS, molecular ecology, nematode, systematics, .

The Internal Transcribed Spacer Region spacer (IGS), and rDNA genes appear to dis- (ITS), located between the repeating array play concerted evolution so that copies of of nuclear 18S and 28S ribosomal DNA these genes from a single individual tend to genes, is a versatile genetic marker. Among be similar to one another, although gener- eukaryotes, including organisms as diverse ally being distinct from those of other spe- as protozoa, plants, vertebrates, and fungi, cies (Elder and Turner, 1995). The applica- ITS data have been used in constructing tion of the ITS to identification has received phylogenetic trees, estimating genetic popu- the most attention by nematologists (Camp- lation structures, evaluating population- bell et al., 1995; Cherry et al., 1997; Chilton level evolutionary processes, and determin- et al., 1995; Epe et al., 1996; Fallas et al., ing taxonomic identity. The structure of the 1996; Ferris et al., 1993, 1994, i995; Gasser rDNA cistron contributes to its wide appli- and Hoste, 1995; Hoste et al., 1995; Ibrahim cability. The rDNA cistron is divided into et al., 1994, 1997;Joyce et al., 1994; Kaplan, domains that evolve at different rates; thus, 1994; Nasmith et al., 1996; Orui, 1996; Reid, this region can be used to address diagnostic 1994; Stevenson et al., 1995; Szalanski et al., and evolutionary problems at different levels 1997; Thiery and Mugniery, 1996; Vrain et of divergence. The rDNA is a component of al., 1992; Vrain and McNamara, 1994; the middle repetitive family of the nuclear Wendt et al., 1995; Zijlstra et al., 1995, DNA genome, and the presence of multiple 1997). The majority of these studies have copies of these genes in the genome facili- focused upon agriculturally important tate PCR amplification from single juvenile plant-parasitic species, parasites, or and adult nematodes. The ITS, intergenic beneficial insect parasites. Yet, to the best of our knoMedge, there is not a single nema-

Received for publication 26 March 1997. tode species that has failed to provide an xJournal Series No. 11958, Agricultural Research Division, amplification product of the ITS region University of Nebraska--Lincoln, and contribution number 98- 1-I of the Kansas Agricultural Experiment Station. when amplified with "universal" PCR Associate Professor, Department of Plant Pathology, 406 primer sets. Universal amplification coupled Plant Sciences Hall, University of Nebraska, Lincoln, NE 68583- 0722. with the ability to amplify ITS from indi- 3 Nematologist, Department of Plant Pathology, Throckmor- vidual nematodes suggests that any species, ton Hall, Kansas State University, Manhattan, KS 66506. 4 Senior Lecturer and Graduate Student, Department of Bi- population, or ecological community of ology, St. Patrick's College, Maynooth, Co. Kildare, Ireland. nematodes can be analyzed using a molecu- Zoologist, Department of Agriculture of Northern Ireland, Newforge Lane, Belfast, United Kingdom. lar approach based on the rDNA ITS region 6 Postdoctoral Research Associate, Graduate Student, and (Vrain and McNamara, 1994). A standard- Research Technologist, Department of Plant Pathology, Uni- versity of Nebraska--Lincoln. ized taxonomic marker would be particu- E-mail: [email protected] larly useful when populations contain a 441 442 Journal of Nematology, Volume 29, No. 4, December 1997 large number of juvenile stages, when sexes This primer set consists of one primer, are dimorphic, or when unfamiliar nema- designated as rDNA2 (5'-TTGATTACGTC- todes are encountered. In ecological stud- CCTGCCCTTT-3'), located in the 3' por- ies, PCR-RFLP profiles of ITS from indi- tion of 18 S, the small ribosomal subunit vidual nematodes may be one method to as- gene, approximately 190 bp from its junc- sess nematode diversity in samples as well as tion with ITS1, the first internally transcribed provide critical taxonomic characters useful spacer. The second primer, designated as for species comparison and identification. rDNA2.144 (5'-GTAGGTGAACCTGCA- In this study we have evaluated the diag- GATGGAT-3'), is located in the 5' portion nostic utility of the ITS region from a wide of 28 S, the large ribosomal subunit gene, taxonomic range of nematodes, including approximately 80 bp from the junction with representatives of both and Ad- ITS2, the second internal transcribed enophorea, among free-living, insect and spacer. Between both spacers is the 5.8 S plant-parasitic species. We demonstrate that ribosomal gene that is generally around 155 the size of the amplified ITS product aids in bp in length. The ITS1 amplification proce- the initial determination of group member- dure in this research used the 18 S primer of ship, and we investigate the occurrence of Vrain et al. (1992), rDNA2, together with a ITS heterogeneity in nematode populations primer located in the first 20 bp of the 5.8 S and individuals and discuss its taxonomic gene flanking ITS1 (Cherry et al., 1997), implications. designated as rDNA1.58s (5'-ACGAGCC- GAGTGATCCACCG-3'). MATERIALS AND METHODS PCR-RFLP was performed according to previously described methods (Cherry et al., Nematode samples: Cephalobid and rhab- 1997; Powers and Harris, 1993). ditid nematodes were obtained from the Caenorhabditis Genetics Center at the Univer- RESULTS AND DISCUSSION sity of Minnesota. Steinernema and Heterorhab- ditis spp. were maintained in the laboratory ITS size variation: The phylum Nemata dis- at the University of Nebraska. plays a wide range of ITS sizes. Figure 1 PCR-RFLP: Amplification of the product shows amplified product of the entire ITS1 including the entire ITS1, 5.8S rDNA gene, and ITS2 region from representative rhab- and ITS2 region was conducted using the ditid and cephalobid nematodes arranged primers described in Vrain et al. (1992). according to size. The estimated size of the

Kb

1.5

1.13

0.5 FIG. l. Amplification of the ITS 1 and 2 regions of rhabditid and cephalobid nematodes. ITS as a Taxonomic Marker: Powers et al. 443 product ranges from 0.7-1.3 kb. Assuming Reptilia, Mammalia) (Fitch et al., 1995). that the flanking 18S and 28S plus the inter- Large genetic distances between Caenorhab- nal 5.8S ribosomal gene sequences comprise ditis spp. also were estimated from a com- approximately 425 bp of that amplified parison of the mitochondrial cytochrome product, the combined size of ITS1 and oxidase subunit II and calmodulin genes ITS2 ranges from 275-875 bp. Slight size (Thomas and Wilson, 1991). The high levels variation is observed among the four Cae- of 18S divergence were presumed to be due norhabditis species; however, ITS size varia- to the ancient origin of nematodes, elevated tion was not detected among seven species rates of molecular evolution, or a combina- of Heterorhabditis (Joyce et al., 1994) or five tion of both factors (Fitch et al., 1995). Like species of Steinernema (T. Powers, unpub- 18S nucleotide divergence, ITS size poly- lished). Rhabditis species include many ITS morphism (Fig. 1) appears to reflect a high size classes (Fig. 1). The existence of size level of genetic divergence among rhabditid variation among rhabditid genera appears and cephalobid nematodes, although the to be correlated with high levels of genetic magnitude of the size difference does not divergence among members of this group. necessarily correspond in a linear fashion to Nucleotide sequence variation of the 18S the degree of divergence based on 18S gene measured among some of the same analysis. rhabditid genera (Fig. 1) were estimated at An equally large range of size variation is eight times the level of divergence observed observed among the plant-parasitic nema- among tetrapod classes (Aves, Amphibia, todes. The ITS1 amplification products

I

A B

• . ". . ! " .

bo ~.° ~.~. ~. ~.~. ~." o.~0 • O. ~" c~ h~

C

D FIG. 2. A) ITS1 size variation among various tylenchid and dorylaimid nematodes. B) ITS1 size variation within Belonolaimidae and Hoplolaimidae. C) ITS1 size variation within . D) ITS1 size variation among Meloidogyne, Nacobbus, and Pratylenchus species. 444 Journal of Nematology, Volume 29, No. 4, December 1997

1 60 m. arenaria TTTGATGGAA ACCAATTTAA TCGCAGTGGC TTGAACCGGG CAAAAGTCGT AACAAGGTAG M. incognita M. javanlca M. chitwoodi M. hapla

61 120

m. arenaria CTGTAGGTGA ACCTGCTGCT GGATCATTAC --TTTATGTG ATGTTC--AA ATTTGAATT-

M. incognita --...... --...... -

M. javanlca --...... --...... -

M. chitwoodi ...... C --T ...... AA.T T ...... A

M. hapla ...... C --T..- ...... --. .... C...-.A

121 180

M. arenaria --CGCAA-TG AAATGAT--C GTTGTGAAAC GGCTGTCGCT GGTGTCTAAG TGTTGCTGAT

M. incogni ta --. .... - ...... --......

M. javanlca --. .... - ...... --...... C ......

M. chi twoodi TA .... C--- ..T.- ...... G .... T ...... C ...... G ......

M. hapla GT.T...--- CGT.T--AT ...... - ...... G ......

181 240

M, arenaria ACGGTTGTGA ACGTCCGTGG CTGTATATGT GGTGACATGT TAGGACTCT ...... AAT M. incognita ...... - ...... M. javanzca ...... - ......

M. chitwoodi T.A ...... G ...... A ...A ..... A ...... T..CT TTATAAG... M. hapla T.R.C...C ...... A ..... A ...... --.CT T ......

241 300

m. arenaria -GAG-TTAAG ACCTAATGAG CCTCTTAAGT GAGGCCGCCA GCAACCTTTT TTT-TCTCTA M. incognita -...- ...... - ...... - ...... M. javanmca -...- ...... - ...... - ......

M. chitwoodi C.----...... T ...... A ...... C..A ...... CA-AA.. M. hapla C.G.T ...... T ...... A ...... TA ...... CA-A...

301 360

M. arenaria CA---TTTTA AAAAAAAA ..... ACTAAAA TTCTACCCTT ATCGGTGGAT CACTAGGCTC M. incognita

M. javanzca . .---...... - ...... T ......

M. chitwoodi A.TTT .... T T.TTC...- .... .AAT ..... T..G ..... C ...... C..T..

M. hapla ---TT .... T ..---...- ...... G ...... T..T ...... C .....

361 420 M. arenaria GTGGATCGAT GAAGAACGCA GCAAACTGCG ATAATTATTG CGAACTGCAG AAGTATTGAG M. incognita M. javanica

M. chitwoodi ...... T ...... TA.. T ...... A.C .....

M. hapla ...... C ...... T ...... TG ...... AC ......

421 480 M. arenaria CACAAAAGTT TTGAACGCAA ATGGCCGCAT TGAGGTCAAA CTCTTTGCAA CGTCTGGTTC M. incognita M. javanica

M. chitwoodi • .T ...... T ..T..G ...... G ...... C ..... GC ......

M. hapla • .T ...... T ..... CT..G,..C ..G...AG,. .C ...... C ...... FIG. 3. Alignment of 18S, ITS1, 5.8S, ITS2, and 28S sequences among five Meloidogyne species. Sequence identity is indicated by ".", deletions by "-", and coding regions are italicized. from 56 nematode species were examined 18 S and 5.8 S ribosomal gene sequences, is (Fig. 2A-D). The 1.2-kb product of Trichod- approximately 215 bp (Fig. 3). This is o~s is three times the size of the 0.4-kb among the shortest ITS1 sequences known Meloidogyne product (Figs. 2B,2D). The ITS1 for any eukaryotic organism. size of Meloidogyne, exclusive of the flanking Congeneric species often display the same ITS as a Taxonomic Marker: Powers et al. 445

481 540 M. arenaria AGGGTCATTT TCTCTTATAG CGGAAGCTTT AATTTCTATA A-TGATGTTG T---T---GC

M. incognita ...... - ...... ---.---..

M. javanica ...... - ...... ---.---..

M. chitwoodi ...... TAC.A .... A ...... T.A..T .... T-..T-A..- ATTG.ATAC.

M. hapla ...... TCTA..A.. TAT..--A.. TTA..T...T T-..CCA..- ...... G.. 541 600 M. arenaria TTTATA ...... TTTTAAAA-GGATT ...... TTTGTTTA .... TT--CA TGTATTAAAT

M. incognita ...... - ...... - ..... - ...... ----..--......

M. javanica ...... - ...... - ..... - ...... ----..--......

M. chitwoodi ...... A ...... -TTC -T.T..TGA- TGCAAT .... - ...... T ..A..- ....

M. hapla AC .... A--- AC ...... TG TT.G.ACGCA GCGA.T.G.. - ...... A. ..A..-..C. 601 660 M. arenaria CTAACTGTGA AAATCAAACA A---TTTTGA CCTGAACTCA GTCGAGAGCA CCCGCTGAAC

M. incognita ...... ---......

M. javanica ...... ---......

M. chitwoodi A.TTG.A.C- ....ATGCTT TATT ...... T ...... M. hapla ..TT---.CG CTG...C.TT TA-T ...... G ...... T ...... 661 704

M. arenaria TTAAGCATAT CAGTAAGCGG AGGAAAAGAA ACTAAATAGG ATTC

M. incognita ......

M. javanica ......

M. chitwoodi ...... C ......

M. hapla ......

GenBank Accession Numbers: M. arenaria U96301, M. incognita

U96304, M. javanica U96305, M. chitwoodi U96302, M. hapla

U96303. FIG. 3. Continued size amplification product. Three many species of nematodes that are difficult spp. (Fig. 3) show a consistent ITS1 size that to identify morphologically. Meloidogyne is slightly larger than that of Globodera, Punc- hapla and M. chitwoodi can be distinguished todera, and Cactodera. Little or no size varia- by digestion of their ITS products with a tion was observed among congeneric species number of restriction enzymes (Zijlstra et of the following genera: Heterodera, Glo- al., 1995). Meloidogynefallax, a species closely bodera, Meloidogyne, Hoplolaimus, , related to M. chitwoodi, can be identified by Belonolaimus, Trichodorus, and Xiphinema. a small length variation in digestion prod- Both Tylenchorhynchus and Pratylenchus have ucts (Zijlstra et al., 1995). Differentiation species displaying extreme ITS1 size varia- among M. arenaria, M. javanica, and M. in- tion (Figs. 2B,2D). ITS size variants have cognita by means of PCR-RFLP profiles has been observed among Aphelenchoides (Ibra- not been achieved (Xue et al., 1993; Zijlstra him et al., 1994) and Pratylenchus (Orui, et al., 1995), and identical copies of the ITS 1996) spp. When large size variation does region have been cloned and sequenced occur in a genus, it may signal the need for from these three Meloidogyne species (Figure taxonomic action. ITS size alone, however, 3). This sequence identity among the three is insufficient evidence upon which to base major mitotic parthenogenetic species is in taxonomic reevaluation. sharp contrast to sequence divergence ob- ITS restriction site variation: Restriction served when those species are compared analyses of PCR-amplified rDNA ITS prod- with M. hapla or M. chitwoodi. Approximately ucts have been used in the diagnosis of 16% sequence divergence is found in these 446 Journal of Nematology, Volume 29, No. 4, December 1997

bp Steinernema carpocapsae All strain 30O

200

100

ranf1 A

Steinernema carpocapsae bp Mexico Kapow UK Agrioto All

300

200

I00

mnfI B FIG. 4. A) Hinf I digest of ITS1 amplicon from Steinernema carpocapsae All strain. B) Hinf I digest of ITS1 amplicon from Steinernema carpocapsae Mexico, Kapow, UK, Agrioto, and All strains.

comparisons, underscoring the large num- the restriction enzyme Fok I (Szalanski et al., ber of discriminating restriction sites avail- 1997). Globodera paUida and G. rostochiensis able for identification of those species by ITS can be separated by several restriction PCR-RFLP. enzymes; however, identification is compli- Globodera and Heterodera species have been cated if G. "mexicana" and G. tabacum iso- examined by PCR-RFLP and nucleotide se- lates are included in comparisons (Thiery quencing (Ferris et al., 1993, 1994, 1995; and Mugniery, 1996). French, U.S., and Thiery and Mugniery, 1996; Szalanski et al., Mexican isolates of G. tabacum produced ITS 1997). The relatively low level of ITS se- profiles very similar to those of G. rostochien- quence divergence between H. glycines and sis, whereas isolates identified as G. "mexi- H. schachtii, reported as less than 1.0% dis- cana"were similar to G. pallida. Each of the similarity (Ferris et al., 1993), is still of suf- four taxa could be discriminated by at least ficient magnitude to allow discrimination by one restriction pattern (Thiery and Mugni- ITS as a Taxonomic Marker: Powers et al. 447 ery, 1996). Laboratory cross-mating between ditis, hermaphroditic reproduction by first- G. pallida and G. rostochiensis has demon- generation adult females. strated that composite ITS restriction pat- Animal-parasitic nematodes for which ITS terns can be produced in hybrid offspring, has shown to be a useful diagnostic tool in- further complicating interpretation of spe- elude Dictyocaulus (Epe et al., 1996), Hy- cies status based on ITS restriction patterns podontus (Chilton et al., 1995), Trichostrongy- alone (Thiery and Mugniery, 1996). lus (Gasser and Hoste, 1995; Hoste et al., Interspecific ITS comparisons in Radopho- 1995), Strongylus (Campbell et al., 1995), lus have been conducted in R. similis, R. ci- and Haemonchus (Stevenson et al., 1995). trophilus, and R. bridgei (Fallas et al., 1996; ITS profiles may be particularly useful in Kaplan, 1994). Limited variation was ob- large genera in which taxonomic characters served using six restriction enzymes in a sur- are difficult to interpret. Helicotylenchus con- vey of R. similis isolates from banana-growing tained 184 described species in 1991 (Eb- regions around the world, but R. similis was sary, 1991), with 34 North American species readily separated from R. bridgei by PCR- (Nematode Geographical Distribution Com- RFLP (Fallas et al., 1996). Kaplan (1994) se- mittee, 1984). The morphological variability quenced the ITS region of representative and small distinctions among Helicotylenchus isolates of R. similis and R. citrophilus and spp. have practically eliminated dichoto- considered the region too highly conserved mous keys as an option for species identifi- for the development of diagnostic assays. cation and have led to the development of a However, among the seven nucleotide dif- probability-based computer identification ferences recorded between those species in system called NEMISYS (Fortuner, 1993). In the ITS1 region, one substitution is pre- our survey of Helicotylenchus isolates from the dicted to result in a restriction site polymor- Great Plains region of North America, we phism; further evaluation of the ITS1 and have found ITS1 patterns correlated with ITS2 regions of Radopholus could produce a plant hosts such as Hinf I patterns from taxonomic marker of the desired specificity. eight individual nematodes representative Other plant-parasitic nematodes that have of populations from agronomic and native been successfully differentiated by PCR- hosts (Fig. 5A). Patterns A1 and A2 have RFLP of ITS include five species of Di- been observed in more than 30 Helicoty- tylenchus and eight described species of Aph- lenchus isolates from corn, soybeans, sor- elenchoides (Ibrahim et al., 1994; Wendt et al., ghum, and bluegrass lawns throughout east- 1995). Among insect-parasitic genera, both ern Kansas, Nebraska, and South Dakota. In Steinernema (Nasmith et al., 1996; Reid, northeast Nebraska and South Dakota coun- 1994) and Heterorhabditis (Joyce et al., 1994; ties bordering Nebraska, five corn fields Nasmith et al., 1996) species display species- were found with mixtures of patterns A1-A2 specific digestion profiles. At subspeeific lev- and B. Pattern C has been observed only els, remarkable consistency is observed from corn in western Kansas, whereas pat- within isolates of one of these genera as terns D1 and D2 have been recorded from demonstrated by a Hinf I digestion of ITS1 native and agronomic hosts from Florida to amplified from 53 infective juveniles repre- Hawaii. Both of these patterns, together senting five strains of S. carpocapsae (Fig. with pattern E, are the predominant HinfI 4A,B). This same enzyme differentiates patterns found on the remnant, native, tall- many of the described species of Steinernema grass prairies in the Great Plains of North (T. Powers, unpublished). The consistency America. D1 was the only pattern observed in patterns among isolates of Steinernema in samples taken from St. Augustine grass and Heterorhabditis species may be due, in throughout Florida. D1 also has been re- part, to homogeneity resulting from found- corded from saguaro cactus in Arizona, in er effects associated with the infection pro- culture on jade plant at Cornell University, cess, inbreeding by sexual stages in the in- and in pineapple fields in Hawaii. Pattern F fected cadaver, or, in the case of Heterorhab- has been observed from Helicotylenchus spp. 448 Journal of Nematology, Volume 29, No. 4, December 1997

from bluegrass lawns in Brookings and ITS heterogeneity: In addition to size varia- Sioux Falls, South Dakota. tion between species and genera and restric- Species names can be placed only tenta- tion site variation between genera, species, tively on the Helicotylenchus specimens from and populations, nematodes also appear to which these patterns were derived. For ex- commonly possess ITS heterogeneity within ample, pattern F in Figure 5A has been ex- individuals. Heterogeneous individuals, de- clusively associated with a Helicotylenchus spe- fined as nematodes containing more than cies that conforms morphologically to H. di- one ITS pattern in their genome, have been gonicus Perry, 1959 and has been collected reported in Meloidogyne (Zijlstra et al., 1995, for molecular studies from localities previ- 1997), Heterodera (Szalanski et al., 1997), Be- ously identified by Thorne and Malek lonolaimus (Cherry et al., 1997), and have (1968). Similarly, many of the cornfield been demonstrated experimentally in hy- samples contained species that could be bridization studies between G. pallida and G. morphologically identified as H. dihystera rostochiensis (Thiery et al., 1996). Amplifica- (Cobb, 1893) Sher, 1961. Yet, from the PCR- tion of ITS with two size classes within an RFLP patterns, it is clear that many popu- individual could be inferred from two- lations identified as H. dihystera exist as banded amplification patterns of Aphelen- mixtures of genotypes. Since many Helicoty- choides besseyi and A. arachidis (Ibrahim et al., lenchus species are collections of partheno- 1994). Heterogeneity also may be inferred genetic lineages, we believe application of from digestion experiments in which diges- species names is ill-advised before a detailed tion profiles exhibit "extra" fragments rela- study that links morphological variation with tive to a standard profile, without the ex- molecular patterns is conducted. pected alteration of another fragment in the

.~c~~ ,,~

A1 A2 B C D1 D2 E F

4

A

Hae III B FIG. 5. A) Eight Hinf I patterns from individual Hdicotylenchu~ nematodes representative of populations from agronomic and native hosts. B) Hae III digestion of Belonolairaus ITS1 amplified from individuals from Nebraska (NE) and Palm Springs, CA (PS) isolates. Nematode DNA was mixed with PUC 19 vector DNA prior to digestion to evaluate completeness of restriction digestion. ITS as a Taxonomic Marker: Powers et al. 449 putative heterogeneous profile. These extra Cherry, T., A.L. Szalanski, T. C. Todd, and T. O. Powers. 1997. The internal transcribed spacer region of fragments also result in inconsistencies be- Belonolaimus (Nemata: Belonolaimidae). Journal of tween the sum of the estimated size of diges- Nematology 29:23-29. tion products and the size of the undigested Chillon, N. B., R. B. Gasser, and I. Beveridge. 1995. amplification product, as in two ITS1 ampli- Differences in a ribosomal DNA sequence of morpho- logically indistinguishable species within the Hypodon- fications from Belonolaimus individuals (Fig. tus macropi complex (Nematoda: Strongyloidea). Inter- 5B) : the Hae III profile of a nematode from nationalJournal for Parasitology 25:64%651. Pahn Springs, Florida (Lane 2), and the Hae Ebsary, B. A. 1991. Catalog of the Order III profile of a nematode from Nebraska (Nematoda). Ottawa: Research Branch, Agriculture (Lane 6). The Nebraska isolate is character- Canada. Elder, J. F., and B.J. Turner. 1995. Concerted evolu- ized by extra fragments of approximately tion of repetitive DNA sequences in eukaryotes. Quar- 170, 70, 60, and 50 bp. Digestion of plasmid terly Review of Biology 70:297-320. DNA mixed with the ITS1 amplification Epe, C., G. von Samson Himmelstjerna, M. Stoye, product (Fig. 5B, lanes 3,5) demonstrated and T. Schnieder. 1996. Comparative molecular char- acterization of Dictyocaubus viviparus and Dictyocaulus eck- that the additional products were not the erti. Berliner und Mfinchener Tierarztliche Wochen- result of incomplete restriction digestion. schrift 109:227-231. ~rhen plasmid DNA was mixed with either Fallas, G. A., M. L. Hahn, M. Fargette, P. R. Burrows, Belonolaimus isolate, a pattern that repre- andJ. L. Sarah. 1996. Molecular and biochemical diver- sity among isolates of Radopholus spp. from different sents a mixture of each of the individual areas of the world. Journal of Nematology 28:422-430. DNA digestions was produced. Digestion Ferris, V. R.,J. M. Ferris, andJ, Faghihi. 1993. Varia- patterns with extra fragments also can be tion in spacer ribosomal DNA in some cyst-forming spe- observed in comparisons of Radopholus simi- cies of plant-parasitic nematodes. Fundamental and Ap- plied Nematology 16:177-184. Iis isolates (Fallas et al., 1996). As a taxo- Ferris, V. R., J. M. Ferris, J. Faghihi, and A. Ireholm. nomic marker in PCR-RFLP studies, hetero- 1994. Comparisons of isolates of Heterodera avenae using geneity provides added genetic resolution 2-D PAGE protein patterns and ribosomal DNA. Jour- and may contribute information in studies nal of Nematology 26:144-151. of population structure and gene flow. How- Ferris, V. R., L. I. Miller, J. Faghihi, and J. M. Ferris. 1995. Ribosomal DNA comparisons of Globodera from ever, ITS heterogeneity will complicate phy- two continents. Journal of Nematology 27:273-283. logenetic analyses unless questions of DNA Fitch, D. H. A., B. Bugaj-Gaweda, and S. W. Emmons. sequence homology can be addressed (Bald- 1995. 18S ribosomal RNA gene phylogeny for some win et al., 1995). Rhabditidae related to Caenorhabditis. Molecular Biol- ogy and Evolution 12:346-358. Overall, the ITS region may become an Fortuner, R. 1993. The NEMISYS solution to prob- important taxonomic feature in future lems in nematode identification. Pp. 137-163 in R. For- nematode diagnoses. ITS versatility, specific- tuner, ed. Advances in computer methods for system- ity, ease of experimental manipulation, and atic biology':Artificial intelligence, databases, computer vision. Baltimore: Johns Hopkins University Press. growing ITS databases should accelerate its Gasser, R. B., and H. Hoste. 1995. Genetic markers application in nematology. Its usefulness, for closely related parasitic nematodes. Molecular and however, will hinge on a careful evaluation Cellular Probes 9:315-319. of the relationship between ITS genetic Hoste, H., N. B. Chilton, and I. Beveridge. 1995. Dif- variation and traditional taxonomic charac- ferences in the second internal transcribed spacer (ri- bosomal DNA) between five species of Trichostrongylus ters. (Nematoda: Trichoshrongylidae). International Jour- nal for Parasitology 25:75-80. LITERATURE CITED Ibrahim, S. IL, R. N. Perry, P. R. Burrows, and D.J. Hooper. 1994. 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