Nematology 2003 Vol 5(1) 99-111

Molecular identi cation of cyst-forming nematodes(Heteroderidae) from Iran and a phylogeny based

on ITS-rDNA sequences

Zahra TANHA MAAFI 1 Sergei A SUBBOTIN 2curren and Maurice MOENS 34

1 Plant Pests and Diseases Research Institute PO Box 1454-Tehran 19395 Iran2 Institute of Parasitology of the Russian Academy of Science Leninskii Prospect 33 Moscow 117071 Russia

3 Agricultural Research Centre Burg Van Gansberghelaan96 Merelbeke 9820 Belgium4 University of Gent Laboratory for Agrozoology Coupure 555 9000 Gent Belgium

Received 17 April 2002 revised 30 September 2002Accepted for publication24 October 2002

Summary ndash RFLP and sequences of ITS-rDNA of 45 populations of cyst-forming nematodes collected from different parts of Iranwere analysed and identi ed as representatives of 21 species Eight enzymes generated RFLP for all studied populations Comparisonof RFLP pro les and sequences of the ITS regions with published data con rmed the presence of Heterodera avenae H lipjeviH glycines H hordecalis H latipons H schachtii and H trifolii in Iran RFLP patterns and ITS sequences for H elachistaH turcomanica H mothi and C cacti were obtained for the rst time in this study Heterodera humuli H goettingiana H ciH elachista H turcomanica and Cactodera cacti are recorded for the rst time in Iran These results correspond with morphologicaland morphometric identi cation of the populations Several populations were not identi ed at the species level and are attributed toHeterodera sp some of these may correspond to new species Twenty-one new sequences from Iranian cyst-forming nematodes and36 known sequences were used for the phylogenetic analyses The cyst-forming nematodes formed several clades corresponding totheir morphological features Heterodera mothi and H elachista clustered with high support with other Cyperi group species andH turcomanica formed a moderately to highly supported clade with the Humuli group

Keywords ndash diagnostics Heterodera PCR phylogeny RFLP

The rst evidence of the presence of cyst-forming ne-matodes in Iran was reported by Esmailpour and Schaumlffer(1970) who detected Heterodera schachtii Schmidt 1871in samples from sugar beet elds in Khorasan provinceeastern Iran Since then H avenae Wollenweber 1924H carotae Jones 1950 H cruciferae Franklin 1945H lipjevi (Madzhidov 1981) Stelter 1984 H galeop-sidis Goffart 1936 H glycines Ichinohe 1952 H horde-calis Anderson 1975 H iri Mathews 1971 H latiponsFranklin 1969 H mani Mathews 1971 H mothi Khanamp Husain 1965 H oryzae Luc amp Berdon Brizuela1961 H rosi Duggan amp Brennan 1966 and H tri-folii Goffart 1932 have been recorded from differentprovinces and various crops (Talachian et al 1976 Nooriet al 1980 Barooti amp Loof 1990 Hojat-Jalali 1991Mehdikhani Moghadam amp Kheiri 1995 Mehdikhani

curren Corresponding author e-mail ssubbotinclofgovbeCorresponding address Agricultural Research Centre Burg Van Gansberghelaan 96 Merelbeke 9820 Belgium

Moghadam et al 1996 Sturhan 1996 MehdikhaniMoghadam 1998 Pedramfar et al 1998 Tanha Maaet al 1999) The identi cation of H galeopsidis H iriH latipons H mothi and H rosi was made on the basis ofvulval cones without examination of the morphology andmorphometrics of second stage juveniles (J2) (Talachianet al 1976 Noori et al 1980) Further investigationsshowed that some species of the Avenae group were notcorrectly identi ed and needed re-examination (Sturhan1996)

Traditional identi cation of cyst-forming nematodesbased on morphological and morphometric characters ofcysts and J2 is time consumingand demands careful studyto separate the sibling species Developed during recentyears ITS-rDNA diagnostics are a reliable tool for identi- cation of cyst-forming nematodes Comparison of RFLP

copy Koninklijke Brill NV Leiden 2003 99Also available online - wwwbrillnl

Z Tanha Maa et al

pro les and sequences of the ITS-rDNA region of un-known species with those publishedand deposited in Gen-Bank (Ferris et al 1994 1999Thieacutery amp Mugnieacutery 1996Orui 1997 Szalanski et al 1997 Subbotin et al 19992000a b 2001 Eroshenko et al 2001) facilitates fastidenti cation of most species of cyst-forming nematode

The goals of the present study were i) to iden-tify Iranian cyst-forming nematode populations based onRFLP and sequences of the ITS region of rDNA ii) topresent RFLP pro les along with information on the exactsizes of the restriction fragments for Iranian cyst-formingnematode species iii) to analyse the phylogenetic rela-tionship between these species based on their ITS-rDNAsequences

Materials and methods

NEMATODE POPULATIONS

During 1998-2000 about 1200 soil and root sampleswere collected from cultivated and wild plants and soilin natural areas agricultural elds and orchards in dif-ferent provinces of Iran The cysts were extracted by acombination of Cobbrsquos sieving and decanting method andsugar otation methods (Caveness amp Jensen 1955 Dunn1969) For each population vulval cones of several cystswere mounted in glycerine jelly Second stage juvenilesfrom the same cyst were xed in TAF and transferred toglycerine (De Grisse 1969) Remaining cysts were airdried and kept at room temperature for molecular workPrimary identi cation was carried out on the basis of mor-phometrics plus morphological characters of cysts andjuveniles (Mulvey 1972 Mulvey amp Golden 1983 Gol-den 1986 Wouts amp Baldwin 1998) A total of 45 cyst-forming nematode populations belonging to 21 specieswere collected (Table 1)

DNA EXTRACTION

Several cysts of each populationwere soaked overnightin double distilled water One to four cysts were putin 8 sup1l ddH2O on a glass slide and punctured under adissecting microscope Second stage juveniles and eggswere transferred to an Eppendorf tube containing 12 sup1lworm lysis buffer (500 mM KCl 100 mM Tris-Cl pH 815 mM MgCl2 10 mM DTT 45 Tween 20) andcrushed with a microhomogeniser Vibro Mixer (ZuumlrichSwitzerland) Two microlitres proteinase K (600 sup1gml)(Promega Benelux Leiden The Netherlands) were added

and the tubes were frozen at iexcl80plusmnC for at least 10 min andthen incubated at 65plusmnC (1 h) and 95plusmnC (10 min) consec-utively After incubation the tubes were centrifuged for2 min at 14 000 rpm and kept at iexcl20plusmnC until use

PCR

Two sup1l of extracted DNA was transferred to an Eppen-dorf tube containing 25 sup1l 10X Taq incubation buffer5 sup1l Q solution 05 sup1l dNTPs mixture (Taq PCR CoreKit Qiagen Hilden Germany) 015 sup1l of each primer(10 sup1gsup1l) (synthesised by Life Technologies Merel-beke Belgium) 02 sup1l Taq Polymerase and double dis-tilled water to a nal volume of 25 sup1l The forward primerTW81 (50-GTTTCCGTAGGTGAACCTGC-3 0) and thereverse primer AB28 (50-ATATGCTTAAGTTCAGCGG-GT-30) as described by Joyce et al (1994) were used inPCR The PCR ampli cation pro le consisted of 4 minat 94plusmnC 35 cycles of 1 min at 94plusmnC 15 min at 55plusmnCand 2 min at 72plusmnC followed by a nal step of 10 minat 72plusmnC Two sup1l of the PCR product was run on a 08TBE buffered agarose gel (100 V 40 min) The remainingPCR product was stored at iexcl20plusmnC until use

RFLP

The PCR product was puri ed using the QIAquickGel Extraction Kit (Qiagen) 2-8 sup1l of puri ed DNAwas digested by one of following restriction enzymesAluI AvaI Bsh1236I BsuRI CfoI MvaI PstI or RsaIin the buffer stipulated by the manufacturer The PCR-product of the Avenae group populations was also re-stricted by HinfI or TaqI to obtain higher discriminationThe digested DNA was run on a 15 TBE bufferedagarose gel stained with ethidium bromide visualisedon UV transilluminator photographed and analysed us-ing Kodak Scienti c Imaging Systems (Kodak RochesterNY USA) The exact length of each restriction frag-ment from the PCR products was obtained by a vir-tual digestion of the sequences using WebCutter 20(www rstmarketcomcuttercut2html)

CLONING AND SEQUENCING

As the initial direct sequences of Heterodera goettin-gianaLiebscher 1892 H humuli Filipjev1934 H mothiH schachtii H trifolii H turcomanica Kirjanova amp Sha-galina 1965 and Heterodera spp 3 4 and 7 showed someambiguous positions the PCR products of these specieswere cloned and re-sequenced DNA was excised from

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Table 1 List of cyst nematode species and populations used in present study

Species Province and locality Plant host Study

Heterodera avenae Ilam Mehran-Reza Abad Wheat (Triticum sp) Sequencing (AF498378) RFLP includingHinfI TaqI

H elachista Mazandaran Tonekabon Rice (Oryza sativa) Sequencing (AF498391) RFLPSoleyman Abad

H ci Kordestan Sanandaj Ficus elastica Sequencing (AF498385) RFLPH lipjevi Fars Abaadeh Tashak Wheat (Triticum sp) RFLP including HinfI TaqI

Kordestan Ghorveh Wheat (Triticum sp) RFLP with HinfI PstI TaqIWestern Azarbayejan Salmas Wheat (Triticum sp) RFLP with HinfI PstI TaqIYazd Marvast Wheat (Triticum sp) RFLP with HinfI PstI TaqIEsfahan Wheat (Triticum sp) Sequencing (AF498380) RFLP with

HinfI PstI TaqIHamadan Kaboutar Ahang Wheat (Triticum sp) RFLP with HinfI PstI TaqIGolestan Gonbad Kavous Wheat (Triticum sp) RFLP with HinfI PstI TaqIMazandaran Sari Sourbon Wheat (Triticum sp) RFLP with HinfI PstI TaqIKerman Bardsir Wheat (Triticum sp) RFLP with HinfI PstI TaqIEastern Azarbayejan Marand Wheat (Triticum sp) RFLP

H glycines Golestan Baghoo Kenareh Soybean (Glycines max) Sequencing (AF498387) RFLPMazandaran Dashtenaz Soybean (Glycines max) RFLP

H goettingiana Lorestan Doroud Unknown RFLPLorestan Doroud Akbar Abad Clover (Trifolium repens) Sequencing (AF498374) RFLP

H hordecalis Ardabil Meshkin shahr Wheat eld and grasses Sequencing (AF498381) RFLPH humuli Mazandaran near Amol Nettle (Urtica dioica) Sequencing (AF498384) RFLPH latipons Western Azarbayejan Salmas Cereals eld Sequencing (AF498382) RFLP

Zanjan Abhar Cereals eld RFLPEastern Azarbayejan Marand Cereals eld RFLPLorestan Doroud Cereals eld RFLPGolestan Agh Ghaleh Cereals eld RFLP

H mothi Khuzestan Ahvaz Cyperus sp Sequencing (AF498392) RFLPSystan and Blouchestan Iranshahr Cyperus sp RFLP

H schachtii Fars Marvdasht Sugar beet (Beta vulgaris) Sequencing (AF498389) RFLPKhorasan Chenaran Sugar beet (Beta vulgaris) RFLPWestern Azarbayejan Khoy Sugar beet (Beta vulgaris) RFLPArdabil Ardabil Sugar beet (Beta vulgaris) RFLPEsfahan Ghohab Sugar beet (Beta vulgaris) RFLP

H trifolii Lorestaan Broujerd Clover (Trifolium sp) Sequencing (AF498388) RFLPH turcomanica Ardabil Meshkin shahr Unknown Sequencing (AF498386) RFLPHeterodera sp 1 Gilan Bandar Anzali Grasses Sequencing (AF498379) RFLP including

HinfI TaqIArdabil Meshkin shahr Grasses RFLP including HinfI TaqITehran Gachsar Grasses RFLP including HinfI TaqI

Heterodera sp 2 Mazandaran Nashtaroud Phragmites sp Sequencing (AF498383) RFLPGilan Bandar Anzali Phragmites sp RFLP

Heterodera sp 3 Tehran Nivaran Park Unknown Sequencing (AF498390) RFLPHeterodera sp 4 Ardabil Moghan Phragmites sp Sequencing (AF498373) RFLPHeterodera sp 5 Tehran Dizin Unknown Sequencing (AF498377) RFLPHeterodera sp 6 Mazandaran Khoram-Abad Unknown Sequencing (AF498375) RFLP

Mazandaran Amol Unknown RFLPHeterodera sp 7 Western Azarbayejan Urumieh Unknown Sequencing (AF498376) RFLPCactodera cacti Markazi Mahalat Unknown Sequencing (AF498393) RFLP

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08 TAE buffered agarose gels using the QIAquick GelExtraction Kit (Qiagen) cloned into the pGEM-T vectorand transformed into JM109 High Ef ciency CompetentCells (Promega Leiden The Netherlands) Several clonesof each species were isolated using bluewhite selectionand submitted to PCR with vector primers PCR productsfrom each sample or clone were sequenced using primersTW81 AB28 or internal reverse primer 58SM5 (50-GGCGCAATGTGCATTCGA-30) as described by Zhenget al (2000) with the BigDye Terminator Cycle Sequenc-ing Ready Reaction Kit (PE Biosystems Benelux TheNetherlands) according to the manufacturerrsquos instructionsThe programme used for sequencing reactions was 30 sat 94plusmnC 30 s at 50plusmnC and 3 min 30 s at 60plusmnC repeatedfor 25 cycles The resulting products were puri ed usinga Centri ex Gel Filtration Cartridge (Edge BioSystemsInc Gaithersburg MD USA) Sequences were run on a377 DNA Sequencer (PE Applied Biosystems Warring-ton UK) The sequences obtained have been submitted tothe GenBank database

SEQUENCE AND PHYLOGENETIC ANALYSES

DNA sequences were edited with Chromas 145 align-ed with Clustal X 164 with default options (Thompsonet al 1997) Only sequences of ITS1-58S-ITS2 wereused for phylogenetic analysis The full ITS regionsequences of several species of the genera HeteroderaCactodera Punctodera and Globodera were obtainedfrom the GenBank database (Ferris et al 1999 Subbotinet al 2001) or from published data (Subbotin et al2000b Zheng et al 2000) Meloidodera alni Turkina ampChizhov 1986 and Cryphodera brinkmani Karssen amp vanAelst 1999 were designated as outgroup taxa (Subbotinet al 2001) Several sequences of ITS1 ITS2 or ITS1-58S-ITS2 deposited in GenBank (Szalanski et al 1997Clapp et al 2000 Sui et al unpubl) were also used forcomparison but were not included in the full sequenceand phylogeneticanalysis

Sequence alignment was analysed with an equallyweighted maximum parsimony (MP) method and mini-mum evolution (ME) method using PAUP 40b4a (Swof-ford 1998) We used heuristic search setting with tenreplicates of random taxon addition tree bisection-recon-

nection branch swapping to seek for the most parsimo-nious trees Gaps were treated as missing data For MEanalysis the appropriate substitution model of DNA evo-lution that best tted the data set was determined bythe Akaike Information Criterion with ModelTest 304(Posada amp Crandall 1998) To obtain an estimation ofthe support for each node a bootstrap analysis (100 repli-cates heuristic search and simple addition of sequence)was also performed

Results

RFLP OF THE ITS-RDNA FOR IRANIAN

POPULATIONS OF CYST-FORMING NEMATODES

Ampli cation of the ITS-rDNA region including ank-ing parts of the 18S and 28S genes yielded a single frag-ment varying from 981 bp in Cactodera cacti (Filipjevamp Schuurmans Stekhoven 1941) Krall amp Krall 1978 to1087 bp in H mothi No PCR products were obtainedin the negative control without nematode DNA templateEight enzymesgeneratedRFLP for all studiedpopulations(Figs 1 2) Sizes of restriction fragments generated byeight enzymes are given in Table 2 Additional restrictionof PCR product by enzymes HinfI and TaqI allowed dis-crimination of the species within the Avenae group HinfIdistinguished Heterodera sp 1 (817 187 41 bp) fromH avenae (499 318 191 41 bp) and TaqI clearly differ-entiated H lipjevi from the other Avenae group species(data not shown) Substantial ITS heterogeneity was re-vealed for H schachtii individuals using MvaI Becauseonly a single clonewas sequencedamong several ITS hap-lotypes the sizes of restriction fragments were not calcu-lated for this species For some species the weak addi-tional bands observed in the RFLP pro les were not takeninto account

Intraspeci c variation in RFLP patterns was not re-vealed for the species studied Heterogeneity of the ITSwas observed in H trifolii H turcomanica H avenaeH glycines H schachtii H lipjevi and Heterodera sp 3and sp 6

Fig 1 Restriction fragments of ampli ed ITS regions of Iranian cyst-forming nematodes A Heterodera avenae B H latiponsC H hordecalis D Heterodera sp 1 E Heterodera sp 2 F H lipjevi G H humuli H H turcomanica I H ci J H trifoliiK H schachtii L Heterodera sp 4 M H glycines N Heterodera sp 3 O H elachista (0 100 bp marker U unrestrictedfragment 1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

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Fig 2 Restriction fragments of amplied ITS regions of Iranian cyst-forming nematodes A Heterodera goettingiana B Heteroderasp 5 C Heterodera sp 6 D Heterodera sp 7 E H mothi F Cactodera cacti (0 100 bp marker U unrestricted fragment1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

SEQUENCE ANALYSIS OF THE ITS REGION

The total length of the alignment including 57 se-quences was 1182 bp Sequence divergence for Het-eroderidae ranged from 00-380 for Heteroderinaefrom 00-302 and for Punctoderinae from 16-165as observed by pairwise comparisons of sequences withadjustment for missing data Sequences of Indian andIranian H avenae populations were identical Sequencesof Georgian and Iranian H ci Kirjanova 1954 werealso similar with the exception of one (A) insertion Se-quences of other Iranian species showed some differ-ences from those of same species collected from otherregions Except for the deletioninsertion of A or a sin-gle or several T after (T)n repeats in some sequences thefollowing nucleotide differences were detected betweenpairs of studied populations three for H humuli (se-quence divergence 03) three for H glycines (03) ve for H hordecalis Andersson 1975 (05) six forH goettingiana (06) ten for H schachtii (11) 12for H latipons (13) and 12 for H trifolii (13) Thepopulationsof the same species always clustered togetherexcept for H trifolii (Fig 3) Morphological identi ca-tion of several other species was not well supported bymolecular data these species being considered as Hetero-dera spp The sequence of the unidenti ed Iranian species

showed a high level of similarity with that of some knownspecies The sequence of Heterodera sp 1 from the Ave-nae group differed by a single T deletion after Tn repeatsand by three and one nucleotides from those of the ITSHav1 and Hav3 clones of Chinese H avenae respectively(Zheng et al 2000) The sequences of Heterodera sp 2and H sacchari and of Heterodera sp 4 and H cynodon-tis Shahina amp Maqbool 1989 differed from each other by22 (22) and 19 (20) nucleotides respectively Hete-rodera spp 6 and 7 showed high similarities with H cru-ciferae H carotae and H urticae Cooper 1955 Differ-ences between these species ranged from ve to 13 nu-cleotides

We found high levels of similarity between our se-quences of H schachtii H goettingiana and H horde-calis and sequences of these species published in Gen-bank (Szalanski et al 1997 Clapp et al 2000 Sui etal unpubl) The H glycines sequences used in our studydiffered by several deletions or insertions from known se-quences (Szalanski et al 1997 Sui et al unpubl)

ANALYSES OF PHYLOGENETIC RELATIONSHIPS

WITHIN SOME CYST-FORMING NEMATODES

Twenty-one new and 36 known sequences were used inthe phylogenetic analyses Minimum evolution and max-

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Table 2 Restriction fragment sizes (bp) of rDNA-ITS regions of cyst-forming nematodes based on RFLP and sequence data

SpeciesEnzyme AluI AvaI Bsh1236I BsuRI CfoI MvaI PstI RsaI

Heterodera (1049) 566 483 1049 897 131 21 420 353 176 745 152 108 400 332 276 708 341 729 320avenae 52 24 24 44 22 19

H elachista 338 325 187 1074 319 220 205 552 236 183 324 248 215 778 296 713 361 618 456179 28 17 201 129 79 24 129 94 64

H ci 756 174 105 922 113 883 131 21 550 434 51 585 152 151 688 199 76 384 336 315 561 474147 72

H lipjevi 569 484 1053 901 131 21 422 379 176 749 152 108 402 334 276 712 211 130 708 600 32452 24 44 41 21

H glycines 341 283 192 552 365 124 498 380 142 524 285 197 (732) 421 311 760 196 85 695 346 818 (620) 217166 44 15 21 24 11 146 117 46 6

H goettingiana 304 239 222 1039 837 202 521 324 170 281 267 194 769 270 533 335 171 470 203 130218 56 24 193 104 119 117

H hordecalis 867 172 1039 706 180 131 529 410 52 735 153 108 431 332 276 698 341 1018 2122 24 24 43

H humuli 451 241 175 925 113 886 131 21 447 438 103 586 152 152 148 766 272 385 337 316 748 243 26171 50 148 21

H latipons 484 343 170 1040 886 131 23 531 408 77 734 108 107 410 332 278 697 343 1019 2125 18 24 47 38 6 20

H mothi 291 246 145 1087 430 316 212 350 246 195 354 258 214 801 286 736 351 666 421117 110 109 129 191 81 24 129 71 56

69 5H trifolii 338 282 180 549 364 112 495 379 130 511 282 197 730 420 308 756 196 73 691 334 814 597 217

166 44 15 21 24 11 119 105 73 205 6H turcomanica 347 327 174 924 111 885 129 21 525 486 24 585 151 150 764 (580) 199 699 336 569 (439) 289

174 13 149 72 150 18 9Heterodera sp 1 562 483 1045 893 131 21 416 353 176 741 152 108 396 276 232 704 341 729 316

52 24 24 44 100 22 19Heterodera sp 2 373 311 141 806 278 465 402 129 538 269 179 402 378 150 808 276 743 341 965 119

132 101 26 67 21 98 58 54 42Heterodera sp 3 383 281 180 550 363 112 496 351 130 510 283 197 419 309 146 756 196 73 1025 691 334 814 597 217

166 15 48 24 11 105 46 205 6Heterodera sp 4 441 381 227 1049 899 150 319 247 182 402 166 159 811 238 623 353 73 1028 21

138 79 36 98 88 8424 13 11 44 8

Heterodera sp 5 304 240 222 1038 835 129 74 521 417 76 270 266 144 767 271 532 336 170 589 321 128168 48 38 24 108 87 61

18 59 43Heterodera sp 6 527 239 220 1041 504 335 129 522 325 170 335 268 171 771 270 534 335 172 590 451

37 18 73 24 140 104 23Heterodera sp 7 527 239 220 1041 839 129 73 522 325 170 475 268 171 771 270 534 335 172 590 (450 400)

37 18 24 104 23 321 130Cactodera cacti 552 239 190 706 275 644 337 458 214 151 343 301 185 331 249 198 643 338 924 545 379

72 40 24 145 7 128 75 36 2122

Italics additional fragments ( ) additional restriction fragments with approximate sizes for some populations or cysts

imum parsimony analyses yielded similar basic relation-ships within Heteroderidae (Figs 3 4) The MP and MEtrees differed with respect to relationships within Cac-todera species and species composing clade VI and VCactodera cacti occupied a basal position in the genusHeterodera mothi and H elachista Ohshima 1974 clus-tered with high support with other Cyperi group speciesHeterodera turcomanica formed a moderate to highlysupported clade with the Humuli group

Discussion

Our comparison of RFLP-ITS pro les and sequencesfrom Iranian populationsof cyst-forming nematodes withpublished data (Orui 1997 Szalanski et al 1997 Sub-botin et al 1997 2000a 2001 Clapp et al 2000) con- rms the presence of H avenae H lipjevi H glycinesH hordecalis H latipons H schachtii and H trifolii inIran RFLP patterns and ITS sequences for H elachista

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Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

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Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

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ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

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Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Z Tanha Maa et al

102 Nematology

Cyst nematodes from Iran

08 TAE buffered agarose gels using the QIAquick GelExtraction Kit (Qiagen) cloned into the pGEM-T vectorand transformed into JM109 High Ef ciency CompetentCells (Promega Leiden The Netherlands) Several clonesof each species were isolated using bluewhite selectionand submitted to PCR with vector primers PCR productsfrom each sample or clone were sequenced using primersTW81 AB28 or internal reverse primer 58SM5 (50-GGCGCAATGTGCATTCGA-30) as described by Zhenget al (2000) with the BigDye Terminator Cycle Sequenc-ing Ready Reaction Kit (PE Biosystems Benelux TheNetherlands) according to the manufacturerrsquos instructionsThe programme used for sequencing reactions was 30 sat 94plusmnC 30 s at 50plusmnC and 3 min 30 s at 60plusmnC repeatedfor 25 cycles The resulting products were puri ed usinga Centri ex Gel Filtration Cartridge (Edge BioSystemsInc Gaithersburg MD USA) Sequences were run on a377 DNA Sequencer (PE Applied Biosystems Warring-ton UK) The sequences obtained have been submitted tothe GenBank database

SEQUENCE AND PHYLOGENETIC ANALYSES

DNA sequences were edited with Chromas 145 align-ed with Clustal X 164 with default options (Thompsonet al 1997) Only sequences of ITS1-58S-ITS2 wereused for phylogenetic analysis The full ITS regionsequences of several species of the genera HeteroderaCactodera Punctodera and Globodera were obtainedfrom the GenBank database (Ferris et al 1999 Subbotinet al 2001) or from published data (Subbotin et al2000b Zheng et al 2000) Meloidodera alni Turkina ampChizhov 1986 and Cryphodera brinkmani Karssen amp vanAelst 1999 were designated as outgroup taxa (Subbotinet al 2001) Several sequences of ITS1 ITS2 or ITS1-58S-ITS2 deposited in GenBank (Szalanski et al 1997Clapp et al 2000 Sui et al unpubl) were also used forcomparison but were not included in the full sequenceand phylogeneticanalysis

Sequence alignment was analysed with an equallyweighted maximum parsimony (MP) method and mini-mum evolution (ME) method using PAUP 40b4a (Swof-ford 1998) We used heuristic search setting with tenreplicates of random taxon addition tree bisection-recon-

nection branch swapping to seek for the most parsimo-nious trees Gaps were treated as missing data For MEanalysis the appropriate substitution model of DNA evo-lution that best tted the data set was determined bythe Akaike Information Criterion with ModelTest 304(Posada amp Crandall 1998) To obtain an estimation ofthe support for each node a bootstrap analysis (100 repli-cates heuristic search and simple addition of sequence)was also performed

Results

RFLP OF THE ITS-RDNA FOR IRANIAN

POPULATIONS OF CYST-FORMING NEMATODES

Ampli cation of the ITS-rDNA region including ank-ing parts of the 18S and 28S genes yielded a single frag-ment varying from 981 bp in Cactodera cacti (Filipjevamp Schuurmans Stekhoven 1941) Krall amp Krall 1978 to1087 bp in H mothi No PCR products were obtainedin the negative control without nematode DNA templateEight enzymesgeneratedRFLP for all studiedpopulations(Figs 1 2) Sizes of restriction fragments generated byeight enzymes are given in Table 2 Additional restrictionof PCR product by enzymes HinfI and TaqI allowed dis-crimination of the species within the Avenae group HinfIdistinguished Heterodera sp 1 (817 187 41 bp) fromH avenae (499 318 191 41 bp) and TaqI clearly differ-entiated H lipjevi from the other Avenae group species(data not shown) Substantial ITS heterogeneity was re-vealed for H schachtii individuals using MvaI Becauseonly a single clonewas sequencedamong several ITS hap-lotypes the sizes of restriction fragments were not calcu-lated for this species For some species the weak addi-tional bands observed in the RFLP pro les were not takeninto account

Intraspeci c variation in RFLP patterns was not re-vealed for the species studied Heterogeneity of the ITSwas observed in H trifolii H turcomanica H avenaeH glycines H schachtii H lipjevi and Heterodera sp 3and sp 6

Fig 1 Restriction fragments of ampli ed ITS regions of Iranian cyst-forming nematodes A Heterodera avenae B H latiponsC H hordecalis D Heterodera sp 1 E Heterodera sp 2 F H lipjevi G H humuli H H turcomanica I H ci J H trifoliiK H schachtii L Heterodera sp 4 M H glycines N Heterodera sp 3 O H elachista (0 100 bp marker U unrestrictedfragment 1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

Vol 5(1) 2003 103

Z Tanha Maa et al

Fig 2 Restriction fragments of amplied ITS regions of Iranian cyst-forming nematodes A Heterodera goettingiana B Heteroderasp 5 C Heterodera sp 6 D Heterodera sp 7 E H mothi F Cactodera cacti (0 100 bp marker U unrestricted fragment1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

SEQUENCE ANALYSIS OF THE ITS REGION

The total length of the alignment including 57 se-quences was 1182 bp Sequence divergence for Het-eroderidae ranged from 00-380 for Heteroderinaefrom 00-302 and for Punctoderinae from 16-165as observed by pairwise comparisons of sequences withadjustment for missing data Sequences of Indian andIranian H avenae populations were identical Sequencesof Georgian and Iranian H ci Kirjanova 1954 werealso similar with the exception of one (A) insertion Se-quences of other Iranian species showed some differ-ences from those of same species collected from otherregions Except for the deletioninsertion of A or a sin-gle or several T after (T)n repeats in some sequences thefollowing nucleotide differences were detected betweenpairs of studied populations three for H humuli (se-quence divergence 03) three for H glycines (03) ve for H hordecalis Andersson 1975 (05) six forH goettingiana (06) ten for H schachtii (11) 12for H latipons (13) and 12 for H trifolii (13) Thepopulationsof the same species always clustered togetherexcept for H trifolii (Fig 3) Morphological identi ca-tion of several other species was not well supported bymolecular data these species being considered as Hetero-dera spp The sequence of the unidenti ed Iranian species

showed a high level of similarity with that of some knownspecies The sequence of Heterodera sp 1 from the Ave-nae group differed by a single T deletion after Tn repeatsand by three and one nucleotides from those of the ITSHav1 and Hav3 clones of Chinese H avenae respectively(Zheng et al 2000) The sequences of Heterodera sp 2and H sacchari and of Heterodera sp 4 and H cynodon-tis Shahina amp Maqbool 1989 differed from each other by22 (22) and 19 (20) nucleotides respectively Hete-rodera spp 6 and 7 showed high similarities with H cru-ciferae H carotae and H urticae Cooper 1955 Differ-ences between these species ranged from ve to 13 nu-cleotides

We found high levels of similarity between our se-quences of H schachtii H goettingiana and H horde-calis and sequences of these species published in Gen-bank (Szalanski et al 1997 Clapp et al 2000 Sui etal unpubl) The H glycines sequences used in our studydiffered by several deletions or insertions from known se-quences (Szalanski et al 1997 Sui et al unpubl)

ANALYSES OF PHYLOGENETIC RELATIONSHIPS

WITHIN SOME CYST-FORMING NEMATODES

Twenty-one new and 36 known sequences were used inthe phylogenetic analyses Minimum evolution and max-

104 Nematology

Cyst nematodes from Iran

Table 2 Restriction fragment sizes (bp) of rDNA-ITS regions of cyst-forming nematodes based on RFLP and sequence data

SpeciesEnzyme AluI AvaI Bsh1236I BsuRI CfoI MvaI PstI RsaI

Heterodera (1049) 566 483 1049 897 131 21 420 353 176 745 152 108 400 332 276 708 341 729 320avenae 52 24 24 44 22 19

H elachista 338 325 187 1074 319 220 205 552 236 183 324 248 215 778 296 713 361 618 456179 28 17 201 129 79 24 129 94 64

H ci 756 174 105 922 113 883 131 21 550 434 51 585 152 151 688 199 76 384 336 315 561 474147 72

H lipjevi 569 484 1053 901 131 21 422 379 176 749 152 108 402 334 276 712 211 130 708 600 32452 24 44 41 21

H glycines 341 283 192 552 365 124 498 380 142 524 285 197 (732) 421 311 760 196 85 695 346 818 (620) 217166 44 15 21 24 11 146 117 46 6

H goettingiana 304 239 222 1039 837 202 521 324 170 281 267 194 769 270 533 335 171 470 203 130218 56 24 193 104 119 117

H hordecalis 867 172 1039 706 180 131 529 410 52 735 153 108 431 332 276 698 341 1018 2122 24 24 43

H humuli 451 241 175 925 113 886 131 21 447 438 103 586 152 152 148 766 272 385 337 316 748 243 26171 50 148 21

H latipons 484 343 170 1040 886 131 23 531 408 77 734 108 107 410 332 278 697 343 1019 2125 18 24 47 38 6 20

H mothi 291 246 145 1087 430 316 212 350 246 195 354 258 214 801 286 736 351 666 421117 110 109 129 191 81 24 129 71 56

69 5H trifolii 338 282 180 549 364 112 495 379 130 511 282 197 730 420 308 756 196 73 691 334 814 597 217

166 44 15 21 24 11 119 105 73 205 6H turcomanica 347 327 174 924 111 885 129 21 525 486 24 585 151 150 764 (580) 199 699 336 569 (439) 289

174 13 149 72 150 18 9Heterodera sp 1 562 483 1045 893 131 21 416 353 176 741 152 108 396 276 232 704 341 729 316

52 24 24 44 100 22 19Heterodera sp 2 373 311 141 806 278 465 402 129 538 269 179 402 378 150 808 276 743 341 965 119

132 101 26 67 21 98 58 54 42Heterodera sp 3 383 281 180 550 363 112 496 351 130 510 283 197 419 309 146 756 196 73 1025 691 334 814 597 217

166 15 48 24 11 105 46 205 6Heterodera sp 4 441 381 227 1049 899 150 319 247 182 402 166 159 811 238 623 353 73 1028 21

138 79 36 98 88 8424 13 11 44 8

Heterodera sp 5 304 240 222 1038 835 129 74 521 417 76 270 266 144 767 271 532 336 170 589 321 128168 48 38 24 108 87 61

18 59 43Heterodera sp 6 527 239 220 1041 504 335 129 522 325 170 335 268 171 771 270 534 335 172 590 451

37 18 73 24 140 104 23Heterodera sp 7 527 239 220 1041 839 129 73 522 325 170 475 268 171 771 270 534 335 172 590 (450 400)

37 18 24 104 23 321 130Cactodera cacti 552 239 190 706 275 644 337 458 214 151 343 301 185 331 249 198 643 338 924 545 379

72 40 24 145 7 128 75 36 2122

Italics additional fragments ( ) additional restriction fragments with approximate sizes for some populations or cysts

imum parsimony analyses yielded similar basic relation-ships within Heteroderidae (Figs 3 4) The MP and MEtrees differed with respect to relationships within Cac-todera species and species composing clade VI and VCactodera cacti occupied a basal position in the genusHeterodera mothi and H elachista Ohshima 1974 clus-tered with high support with other Cyperi group speciesHeterodera turcomanica formed a moderate to highlysupported clade with the Humuli group

Discussion

Our comparison of RFLP-ITS pro les and sequencesfrom Iranian populationsof cyst-forming nematodes withpublished data (Orui 1997 Szalanski et al 1997 Sub-botin et al 1997 2000a 2001 Clapp et al 2000) con- rms the presence of H avenae H lipjevi H glycinesH hordecalis H latipons H schachtii and H trifolii inIran RFLP patterns and ITS sequences for H elachista

Vol 5(1) 2003 105

Z Tanha Maa et al

Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

106 Nematology

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Cyst nematodes from Iran

08 TAE buffered agarose gels using the QIAquick GelExtraction Kit (Qiagen) cloned into the pGEM-T vectorand transformed into JM109 High Ef ciency CompetentCells (Promega Leiden The Netherlands) Several clonesof each species were isolated using bluewhite selectionand submitted to PCR with vector primers PCR productsfrom each sample or clone were sequenced using primersTW81 AB28 or internal reverse primer 58SM5 (50-GGCGCAATGTGCATTCGA-30) as described by Zhenget al (2000) with the BigDye Terminator Cycle Sequenc-ing Ready Reaction Kit (PE Biosystems Benelux TheNetherlands) according to the manufacturerrsquos instructionsThe programme used for sequencing reactions was 30 sat 94plusmnC 30 s at 50plusmnC and 3 min 30 s at 60plusmnC repeatedfor 25 cycles The resulting products were puri ed usinga Centri ex Gel Filtration Cartridge (Edge BioSystemsInc Gaithersburg MD USA) Sequences were run on a377 DNA Sequencer (PE Applied Biosystems Warring-ton UK) The sequences obtained have been submitted tothe GenBank database

SEQUENCE AND PHYLOGENETIC ANALYSES

DNA sequences were edited with Chromas 145 align-ed with Clustal X 164 with default options (Thompsonet al 1997) Only sequences of ITS1-58S-ITS2 wereused for phylogenetic analysis The full ITS regionsequences of several species of the genera HeteroderaCactodera Punctodera and Globodera were obtainedfrom the GenBank database (Ferris et al 1999 Subbotinet al 2001) or from published data (Subbotin et al2000b Zheng et al 2000) Meloidodera alni Turkina ampChizhov 1986 and Cryphodera brinkmani Karssen amp vanAelst 1999 were designated as outgroup taxa (Subbotinet al 2001) Several sequences of ITS1 ITS2 or ITS1-58S-ITS2 deposited in GenBank (Szalanski et al 1997Clapp et al 2000 Sui et al unpubl) were also used forcomparison but were not included in the full sequenceand phylogeneticanalysis

Sequence alignment was analysed with an equallyweighted maximum parsimony (MP) method and mini-mum evolution (ME) method using PAUP 40b4a (Swof-ford 1998) We used heuristic search setting with tenreplicates of random taxon addition tree bisection-recon-

nection branch swapping to seek for the most parsimo-nious trees Gaps were treated as missing data For MEanalysis the appropriate substitution model of DNA evo-lution that best tted the data set was determined bythe Akaike Information Criterion with ModelTest 304(Posada amp Crandall 1998) To obtain an estimation ofthe support for each node a bootstrap analysis (100 repli-cates heuristic search and simple addition of sequence)was also performed

Results

RFLP OF THE ITS-RDNA FOR IRANIAN

POPULATIONS OF CYST-FORMING NEMATODES

Ampli cation of the ITS-rDNA region including ank-ing parts of the 18S and 28S genes yielded a single frag-ment varying from 981 bp in Cactodera cacti (Filipjevamp Schuurmans Stekhoven 1941) Krall amp Krall 1978 to1087 bp in H mothi No PCR products were obtainedin the negative control without nematode DNA templateEight enzymesgeneratedRFLP for all studiedpopulations(Figs 1 2) Sizes of restriction fragments generated byeight enzymes are given in Table 2 Additional restrictionof PCR product by enzymes HinfI and TaqI allowed dis-crimination of the species within the Avenae group HinfIdistinguished Heterodera sp 1 (817 187 41 bp) fromH avenae (499 318 191 41 bp) and TaqI clearly differ-entiated H lipjevi from the other Avenae group species(data not shown) Substantial ITS heterogeneity was re-vealed for H schachtii individuals using MvaI Becauseonly a single clonewas sequencedamong several ITS hap-lotypes the sizes of restriction fragments were not calcu-lated for this species For some species the weak addi-tional bands observed in the RFLP pro les were not takeninto account

Intraspeci c variation in RFLP patterns was not re-vealed for the species studied Heterogeneity of the ITSwas observed in H trifolii H turcomanica H avenaeH glycines H schachtii H lipjevi and Heterodera sp 3and sp 6

Fig 1 Restriction fragments of ampli ed ITS regions of Iranian cyst-forming nematodes A Heterodera avenae B H latiponsC H hordecalis D Heterodera sp 1 E Heterodera sp 2 F H lipjevi G H humuli H H turcomanica I H ci J H trifoliiK H schachtii L Heterodera sp 4 M H glycines N Heterodera sp 3 O H elachista (0 100 bp marker U unrestrictedfragment 1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

Vol 5(1) 2003 103

Z Tanha Maa et al

Fig 2 Restriction fragments of amplied ITS regions of Iranian cyst-forming nematodes A Heterodera goettingiana B Heteroderasp 5 C Heterodera sp 6 D Heterodera sp 7 E H mothi F Cactodera cacti (0 100 bp marker U unrestricted fragment1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

SEQUENCE ANALYSIS OF THE ITS REGION

The total length of the alignment including 57 se-quences was 1182 bp Sequence divergence for Het-eroderidae ranged from 00-380 for Heteroderinaefrom 00-302 and for Punctoderinae from 16-165as observed by pairwise comparisons of sequences withadjustment for missing data Sequences of Indian andIranian H avenae populations were identical Sequencesof Georgian and Iranian H ci Kirjanova 1954 werealso similar with the exception of one (A) insertion Se-quences of other Iranian species showed some differ-ences from those of same species collected from otherregions Except for the deletioninsertion of A or a sin-gle or several T after (T)n repeats in some sequences thefollowing nucleotide differences were detected betweenpairs of studied populations three for H humuli (se-quence divergence 03) three for H glycines (03) ve for H hordecalis Andersson 1975 (05) six forH goettingiana (06) ten for H schachtii (11) 12for H latipons (13) and 12 for H trifolii (13) Thepopulationsof the same species always clustered togetherexcept for H trifolii (Fig 3) Morphological identi ca-tion of several other species was not well supported bymolecular data these species being considered as Hetero-dera spp The sequence of the unidenti ed Iranian species

showed a high level of similarity with that of some knownspecies The sequence of Heterodera sp 1 from the Ave-nae group differed by a single T deletion after Tn repeatsand by three and one nucleotides from those of the ITSHav1 and Hav3 clones of Chinese H avenae respectively(Zheng et al 2000) The sequences of Heterodera sp 2and H sacchari and of Heterodera sp 4 and H cynodon-tis Shahina amp Maqbool 1989 differed from each other by22 (22) and 19 (20) nucleotides respectively Hete-rodera spp 6 and 7 showed high similarities with H cru-ciferae H carotae and H urticae Cooper 1955 Differ-ences between these species ranged from ve to 13 nu-cleotides

We found high levels of similarity between our se-quences of H schachtii H goettingiana and H horde-calis and sequences of these species published in Gen-bank (Szalanski et al 1997 Clapp et al 2000 Sui etal unpubl) The H glycines sequences used in our studydiffered by several deletions or insertions from known se-quences (Szalanski et al 1997 Sui et al unpubl)

ANALYSES OF PHYLOGENETIC RELATIONSHIPS

WITHIN SOME CYST-FORMING NEMATODES

Twenty-one new and 36 known sequences were used inthe phylogenetic analyses Minimum evolution and max-

104 Nematology

Cyst nematodes from Iran

Table 2 Restriction fragment sizes (bp) of rDNA-ITS regions of cyst-forming nematodes based on RFLP and sequence data

SpeciesEnzyme AluI AvaI Bsh1236I BsuRI CfoI MvaI PstI RsaI

Heterodera (1049) 566 483 1049 897 131 21 420 353 176 745 152 108 400 332 276 708 341 729 320avenae 52 24 24 44 22 19

H elachista 338 325 187 1074 319 220 205 552 236 183 324 248 215 778 296 713 361 618 456179 28 17 201 129 79 24 129 94 64

H ci 756 174 105 922 113 883 131 21 550 434 51 585 152 151 688 199 76 384 336 315 561 474147 72

H lipjevi 569 484 1053 901 131 21 422 379 176 749 152 108 402 334 276 712 211 130 708 600 32452 24 44 41 21

H glycines 341 283 192 552 365 124 498 380 142 524 285 197 (732) 421 311 760 196 85 695 346 818 (620) 217166 44 15 21 24 11 146 117 46 6

H goettingiana 304 239 222 1039 837 202 521 324 170 281 267 194 769 270 533 335 171 470 203 130218 56 24 193 104 119 117

H hordecalis 867 172 1039 706 180 131 529 410 52 735 153 108 431 332 276 698 341 1018 2122 24 24 43

H humuli 451 241 175 925 113 886 131 21 447 438 103 586 152 152 148 766 272 385 337 316 748 243 26171 50 148 21

H latipons 484 343 170 1040 886 131 23 531 408 77 734 108 107 410 332 278 697 343 1019 2125 18 24 47 38 6 20

H mothi 291 246 145 1087 430 316 212 350 246 195 354 258 214 801 286 736 351 666 421117 110 109 129 191 81 24 129 71 56

69 5H trifolii 338 282 180 549 364 112 495 379 130 511 282 197 730 420 308 756 196 73 691 334 814 597 217

166 44 15 21 24 11 119 105 73 205 6H turcomanica 347 327 174 924 111 885 129 21 525 486 24 585 151 150 764 (580) 199 699 336 569 (439) 289

174 13 149 72 150 18 9Heterodera sp 1 562 483 1045 893 131 21 416 353 176 741 152 108 396 276 232 704 341 729 316

52 24 24 44 100 22 19Heterodera sp 2 373 311 141 806 278 465 402 129 538 269 179 402 378 150 808 276 743 341 965 119

132 101 26 67 21 98 58 54 42Heterodera sp 3 383 281 180 550 363 112 496 351 130 510 283 197 419 309 146 756 196 73 1025 691 334 814 597 217

166 15 48 24 11 105 46 205 6Heterodera sp 4 441 381 227 1049 899 150 319 247 182 402 166 159 811 238 623 353 73 1028 21

138 79 36 98 88 8424 13 11 44 8

Heterodera sp 5 304 240 222 1038 835 129 74 521 417 76 270 266 144 767 271 532 336 170 589 321 128168 48 38 24 108 87 61

18 59 43Heterodera sp 6 527 239 220 1041 504 335 129 522 325 170 335 268 171 771 270 534 335 172 590 451

37 18 73 24 140 104 23Heterodera sp 7 527 239 220 1041 839 129 73 522 325 170 475 268 171 771 270 534 335 172 590 (450 400)

37 18 24 104 23 321 130Cactodera cacti 552 239 190 706 275 644 337 458 214 151 343 301 185 331 249 198 643 338 924 545 379

72 40 24 145 7 128 75 36 2122

Italics additional fragments ( ) additional restriction fragments with approximate sizes for some populations or cysts

imum parsimony analyses yielded similar basic relation-ships within Heteroderidae (Figs 3 4) The MP and MEtrees differed with respect to relationships within Cac-todera species and species composing clade VI and VCactodera cacti occupied a basal position in the genusHeterodera mothi and H elachista Ohshima 1974 clus-tered with high support with other Cyperi group speciesHeterodera turcomanica formed a moderate to highlysupported clade with the Humuli group

Discussion

Our comparison of RFLP-ITS pro les and sequencesfrom Iranian populationsof cyst-forming nematodes withpublished data (Orui 1997 Szalanski et al 1997 Sub-botin et al 1997 2000a 2001 Clapp et al 2000) con- rms the presence of H avenae H lipjevi H glycinesH hordecalis H latipons H schachtii and H trifolii inIran RFLP patterns and ITS sequences for H elachista

Vol 5(1) 2003 105

Z Tanha Maa et al

Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

106 Nematology

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Z Tanha Maa et al

Fig 2 Restriction fragments of amplied ITS regions of Iranian cyst-forming nematodes A Heterodera goettingiana B Heteroderasp 5 C Heterodera sp 6 D Heterodera sp 7 E H mothi F Cactodera cacti (0 100 bp marker U unrestricted fragment1 AluI 2 AvaI 3 Bsh1236I 4 BsuRI 5 CfoI 6 MvaI 7 PstI or 8 RsaI )

SEQUENCE ANALYSIS OF THE ITS REGION

The total length of the alignment including 57 se-quences was 1182 bp Sequence divergence for Het-eroderidae ranged from 00-380 for Heteroderinaefrom 00-302 and for Punctoderinae from 16-165as observed by pairwise comparisons of sequences withadjustment for missing data Sequences of Indian andIranian H avenae populations were identical Sequencesof Georgian and Iranian H ci Kirjanova 1954 werealso similar with the exception of one (A) insertion Se-quences of other Iranian species showed some differ-ences from those of same species collected from otherregions Except for the deletioninsertion of A or a sin-gle or several T after (T)n repeats in some sequences thefollowing nucleotide differences were detected betweenpairs of studied populations three for H humuli (se-quence divergence 03) three for H glycines (03) ve for H hordecalis Andersson 1975 (05) six forH goettingiana (06) ten for H schachtii (11) 12for H latipons (13) and 12 for H trifolii (13) Thepopulationsof the same species always clustered togetherexcept for H trifolii (Fig 3) Morphological identi ca-tion of several other species was not well supported bymolecular data these species being considered as Hetero-dera spp The sequence of the unidenti ed Iranian species

showed a high level of similarity with that of some knownspecies The sequence of Heterodera sp 1 from the Ave-nae group differed by a single T deletion after Tn repeatsand by three and one nucleotides from those of the ITSHav1 and Hav3 clones of Chinese H avenae respectively(Zheng et al 2000) The sequences of Heterodera sp 2and H sacchari and of Heterodera sp 4 and H cynodon-tis Shahina amp Maqbool 1989 differed from each other by22 (22) and 19 (20) nucleotides respectively Hete-rodera spp 6 and 7 showed high similarities with H cru-ciferae H carotae and H urticae Cooper 1955 Differ-ences between these species ranged from ve to 13 nu-cleotides

We found high levels of similarity between our se-quences of H schachtii H goettingiana and H horde-calis and sequences of these species published in Gen-bank (Szalanski et al 1997 Clapp et al 2000 Sui etal unpubl) The H glycines sequences used in our studydiffered by several deletions or insertions from known se-quences (Szalanski et al 1997 Sui et al unpubl)

ANALYSES OF PHYLOGENETIC RELATIONSHIPS

WITHIN SOME CYST-FORMING NEMATODES

Twenty-one new and 36 known sequences were used inthe phylogenetic analyses Minimum evolution and max-

104 Nematology

Cyst nematodes from Iran

Table 2 Restriction fragment sizes (bp) of rDNA-ITS regions of cyst-forming nematodes based on RFLP and sequence data

SpeciesEnzyme AluI AvaI Bsh1236I BsuRI CfoI MvaI PstI RsaI

Heterodera (1049) 566 483 1049 897 131 21 420 353 176 745 152 108 400 332 276 708 341 729 320avenae 52 24 24 44 22 19

H elachista 338 325 187 1074 319 220 205 552 236 183 324 248 215 778 296 713 361 618 456179 28 17 201 129 79 24 129 94 64

H ci 756 174 105 922 113 883 131 21 550 434 51 585 152 151 688 199 76 384 336 315 561 474147 72

H lipjevi 569 484 1053 901 131 21 422 379 176 749 152 108 402 334 276 712 211 130 708 600 32452 24 44 41 21

H glycines 341 283 192 552 365 124 498 380 142 524 285 197 (732) 421 311 760 196 85 695 346 818 (620) 217166 44 15 21 24 11 146 117 46 6

H goettingiana 304 239 222 1039 837 202 521 324 170 281 267 194 769 270 533 335 171 470 203 130218 56 24 193 104 119 117

H hordecalis 867 172 1039 706 180 131 529 410 52 735 153 108 431 332 276 698 341 1018 2122 24 24 43

H humuli 451 241 175 925 113 886 131 21 447 438 103 586 152 152 148 766 272 385 337 316 748 243 26171 50 148 21

H latipons 484 343 170 1040 886 131 23 531 408 77 734 108 107 410 332 278 697 343 1019 2125 18 24 47 38 6 20

H mothi 291 246 145 1087 430 316 212 350 246 195 354 258 214 801 286 736 351 666 421117 110 109 129 191 81 24 129 71 56

69 5H trifolii 338 282 180 549 364 112 495 379 130 511 282 197 730 420 308 756 196 73 691 334 814 597 217

166 44 15 21 24 11 119 105 73 205 6H turcomanica 347 327 174 924 111 885 129 21 525 486 24 585 151 150 764 (580) 199 699 336 569 (439) 289

174 13 149 72 150 18 9Heterodera sp 1 562 483 1045 893 131 21 416 353 176 741 152 108 396 276 232 704 341 729 316

52 24 24 44 100 22 19Heterodera sp 2 373 311 141 806 278 465 402 129 538 269 179 402 378 150 808 276 743 341 965 119

132 101 26 67 21 98 58 54 42Heterodera sp 3 383 281 180 550 363 112 496 351 130 510 283 197 419 309 146 756 196 73 1025 691 334 814 597 217

166 15 48 24 11 105 46 205 6Heterodera sp 4 441 381 227 1049 899 150 319 247 182 402 166 159 811 238 623 353 73 1028 21

138 79 36 98 88 8424 13 11 44 8

Heterodera sp 5 304 240 222 1038 835 129 74 521 417 76 270 266 144 767 271 532 336 170 589 321 128168 48 38 24 108 87 61

18 59 43Heterodera sp 6 527 239 220 1041 504 335 129 522 325 170 335 268 171 771 270 534 335 172 590 451

37 18 73 24 140 104 23Heterodera sp 7 527 239 220 1041 839 129 73 522 325 170 475 268 171 771 270 534 335 172 590 (450 400)

37 18 24 104 23 321 130Cactodera cacti 552 239 190 706 275 644 337 458 214 151 343 301 185 331 249 198 643 338 924 545 379

72 40 24 145 7 128 75 36 2122

Italics additional fragments ( ) additional restriction fragments with approximate sizes for some populations or cysts

imum parsimony analyses yielded similar basic relation-ships within Heteroderidae (Figs 3 4) The MP and MEtrees differed with respect to relationships within Cac-todera species and species composing clade VI and VCactodera cacti occupied a basal position in the genusHeterodera mothi and H elachista Ohshima 1974 clus-tered with high support with other Cyperi group speciesHeterodera turcomanica formed a moderate to highlysupported clade with the Humuli group

Discussion

Our comparison of RFLP-ITS pro les and sequencesfrom Iranian populationsof cyst-forming nematodes withpublished data (Orui 1997 Szalanski et al 1997 Sub-botin et al 1997 2000a 2001 Clapp et al 2000) con- rms the presence of H avenae H lipjevi H glycinesH hordecalis H latipons H schachtii and H trifolii inIran RFLP patterns and ITS sequences for H elachista

Vol 5(1) 2003 105

Z Tanha Maa et al

Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

106 Nematology

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Cyst nematodes from Iran

Table 2 Restriction fragment sizes (bp) of rDNA-ITS regions of cyst-forming nematodes based on RFLP and sequence data

SpeciesEnzyme AluI AvaI Bsh1236I BsuRI CfoI MvaI PstI RsaI

Heterodera (1049) 566 483 1049 897 131 21 420 353 176 745 152 108 400 332 276 708 341 729 320avenae 52 24 24 44 22 19

H elachista 338 325 187 1074 319 220 205 552 236 183 324 248 215 778 296 713 361 618 456179 28 17 201 129 79 24 129 94 64

H ci 756 174 105 922 113 883 131 21 550 434 51 585 152 151 688 199 76 384 336 315 561 474147 72

H lipjevi 569 484 1053 901 131 21 422 379 176 749 152 108 402 334 276 712 211 130 708 600 32452 24 44 41 21

H glycines 341 283 192 552 365 124 498 380 142 524 285 197 (732) 421 311 760 196 85 695 346 818 (620) 217166 44 15 21 24 11 146 117 46 6

H goettingiana 304 239 222 1039 837 202 521 324 170 281 267 194 769 270 533 335 171 470 203 130218 56 24 193 104 119 117

H hordecalis 867 172 1039 706 180 131 529 410 52 735 153 108 431 332 276 698 341 1018 2122 24 24 43

H humuli 451 241 175 925 113 886 131 21 447 438 103 586 152 152 148 766 272 385 337 316 748 243 26171 50 148 21

H latipons 484 343 170 1040 886 131 23 531 408 77 734 108 107 410 332 278 697 343 1019 2125 18 24 47 38 6 20

H mothi 291 246 145 1087 430 316 212 350 246 195 354 258 214 801 286 736 351 666 421117 110 109 129 191 81 24 129 71 56

69 5H trifolii 338 282 180 549 364 112 495 379 130 511 282 197 730 420 308 756 196 73 691 334 814 597 217

166 44 15 21 24 11 119 105 73 205 6H turcomanica 347 327 174 924 111 885 129 21 525 486 24 585 151 150 764 (580) 199 699 336 569 (439) 289

174 13 149 72 150 18 9Heterodera sp 1 562 483 1045 893 131 21 416 353 176 741 152 108 396 276 232 704 341 729 316

52 24 24 44 100 22 19Heterodera sp 2 373 311 141 806 278 465 402 129 538 269 179 402 378 150 808 276 743 341 965 119

132 101 26 67 21 98 58 54 42Heterodera sp 3 383 281 180 550 363 112 496 351 130 510 283 197 419 309 146 756 196 73 1025 691 334 814 597 217

166 15 48 24 11 105 46 205 6Heterodera sp 4 441 381 227 1049 899 150 319 247 182 402 166 159 811 238 623 353 73 1028 21

138 79 36 98 88 8424 13 11 44 8

Heterodera sp 5 304 240 222 1038 835 129 74 521 417 76 270 266 144 767 271 532 336 170 589 321 128168 48 38 24 108 87 61

18 59 43Heterodera sp 6 527 239 220 1041 504 335 129 522 325 170 335 268 171 771 270 534 335 172 590 451

37 18 73 24 140 104 23Heterodera sp 7 527 239 220 1041 839 129 73 522 325 170 475 268 171 771 270 534 335 172 590 (450 400)

37 18 24 104 23 321 130Cactodera cacti 552 239 190 706 275 644 337 458 214 151 343 301 185 331 249 198 643 338 924 545 379

72 40 24 145 7 128 75 36 2122

Italics additional fragments ( ) additional restriction fragments with approximate sizes for some populations or cysts

imum parsimony analyses yielded similar basic relation-ships within Heteroderidae (Figs 3 4) The MP and MEtrees differed with respect to relationships within Cac-todera species and species composing clade VI and VCactodera cacti occupied a basal position in the genusHeterodera mothi and H elachista Ohshima 1974 clus-tered with high support with other Cyperi group speciesHeterodera turcomanica formed a moderate to highlysupported clade with the Humuli group

Discussion

Our comparison of RFLP-ITS pro les and sequencesfrom Iranian populationsof cyst-forming nematodes withpublished data (Orui 1997 Szalanski et al 1997 Sub-botin et al 1997 2000a 2001 Clapp et al 2000) con- rms the presence of H avenae H lipjevi H glycinesH hordecalis H latipons H schachtii and H trifolii inIran RFLP patterns and ITS sequences for H elachista

Vol 5(1) 2003 105

Z Tanha Maa et al

Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

106 Nematology

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Z Tanha Maa et al

Fig 3 Minimum evolution (GTR C I C G model and neighbour-joining method) tree constructed from the ITS sequence alignmentfor 57 species of cyst-forming nematodes Bootstrap values (more than 70) are given in the appropriate clades New sequences areindicated by bold

H turcomanica H mothi and C cacti were obtained forthe rst time in this study Heterodera humuli H goettin-giana H ci H elachista H turcomanica and C cactiare recorded for the rst time in Iran

Identi cation of Iranian cyst-forming nematodes in thiswork has been made based on combinedanalysesof mole-cular (sequence similarity autopomorphic character) re-sults with morphological morphometric (J2 and cysts)

106 Nematology

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Cyst nematodes from Iran

Fig 4 Strict consensus of 24 maximum parsimonious trees constructed from the ITS sequence alignment for 57 species of cyst-formingnematodes Bootstrap values (more than 70) and numbers of nucleotide changes (bold) are given in the appropriate clades (Treelength D 2886 number of parsimony-informative characters D 681 CI D 05274 HI D 04726 RI D 08199 RC D 04324) Newsequences are indicated by bold

and biological (host-plant) data for each populationIf theidenti cation result of any of these approaches could beconsidered as indicating a signi cant difference with thecharacteristics of known species we consideredthis popu-lation as Heterodera sp Seven unidenti ed and possiblynew species were recognised amongst the studied IranianpopulationsFurther comparative taxonomicstudies how-

ever are required to clarify their relationshipswith knowncyst nematodes

Our study con rms the earlier identi cation in Iran ofH lipjevi (Sturhan 1996 Damadzahed amp Ansaripour2001) Although the earlier report of H avenae (Barootiamp Loof 1990 Hojat-Jalali 1991) in Iran was revealed tobe H lipjevi (Sturhan 1996) our study supports the pres-

Vol 5(1) 2003 107

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Z Tanha Maa et al

ence of H avenae and provides additional information onthe variability of RFLP pro les for the latter species gen-erated by AluI This enzyme reveals heterogeneity of theITS region among several specimens from some popula-tions of H avenae These RFLP pro les should be con-sidered for identi cation of populations of this species

Our data further indicate the presence of anotherspecies from the Avenae group Heterodera sp 1 mor-phologically and genetically very similar to H praten-sis Gaumlbler Sturhan Subbotin amp Rumpenhorst 2000 (seeGaumlbler et al 2000) and H avenae from China It wasfound on grasses in three different provinces Howeverbecause of the presence of some minor differences in theITS sequences we could neither attribute these popula-tions to H pratensis nor to the Chinese H avenae be-cause of the difference in host-plants (Subbotin et al un-publ)

The ITS sequences and RFLP pro les of the IranianH latipons populations differ from those for the Rostovpopulation (Ferris et al 1999 Subbotin et al 2000a)and from the sequence of the Syrian population(Subbotinet al 2001) More detailed studies should be conductedto clarify the taxonomic status of the H latipons popula-tions

Heterodera sp 2 was collected on Phragmites sp intwo Iranian provinces These populations are morpho-logically similar to H sacchari Luc amp Merny 1963( nger-like projections on the underbridge many bul-lae three lateral lines stylet knobs deeply forwardlyconcave and three annules in the lip region) Howevermorphometric study showed differences between thesepopulations and H sacchari in body stylet tail andhyaline part of the tail of the second stage juveniles(Tanha Maa et al unpubl) Comparison of the ITS se-quence of one population with the published sequencefor H sacchari (Subbotin et al 2001) revealed rathervariable nucleotide substitutions between them There-fore we consider these populationsas representing a newspecies

A cyst-forming nematode population was collectedfrom roots and soil from the rhizosphere of Urticadioica L in Mazandaran province Comparison of itsRFLP pro le and the ITS sequence of this populationwith published data (Subbotin et al 1997 2000a 2001Eroshenko et al 2001) allowed us to conclude thatthe species was H humuli The presence of numerousnematodes of different developmental stages on the rootsand in the rhizosphere indicates that common nettle is asuitable host for this species

Heterodera turcomanica which was morphometricallyidentical to data taken from the redescription of thisspecies made by Sturhan and Wouts (1995) was found inArdabil province in the northern west part of country TheRFLP and ITS sequence easily distinguish this speciesfrom the morphologically similar H latipons which hasa similar cyst cone structure but lacks an underbridgeAttempts to determine the host plants failed but thefollowing plants were identi ed in the sampling areaSetaria viridis (L) Beauv Daucus carota L Thesiumarvense Horv and Euphorbia boissieriana (Woronow)Prokh

Heterodera trifolii has previously been reported fromIran in sugar beet elds (Talachian et al 1976) In thepresent study a population from clover (Trifolium repensL) was studied In molecular trees it clustered with H ci-ceri Vovlas Greco amp Di Vito 1985 Another populationcollected from the rhizosphere of Oxalis sp and cloverwas initially identi ed morphologically as H trifolii Asit showed sequence dissimilarity with H trifolii and H ci-ceri it is herein regarded as Heterodera sp 3 Phylogeneticand sequenceanalyses of the ITS clones obtainedfrom thegenetically and morphologically closely related speciesH trifolii H ciceri H galeopsidis H daverti Woutsamp Sturhan 1979 and unidenti ed Heterodera popula-tions from Plantago and Rumex failed to distinguish thesespecies and populations and other genes should be usedto discriminate this species complex (Subbotin et al un-publ)

For the differentiation of H elachista and H oryzicolaRao amp Jayaprakesh 1984 both infecting rice Wouts andBaldwin (1998) proposed the shape of stylet knobs of thesecond stage juvenile as a key character whereas Nobbset al (1992) indicated differences in morphometricsand esterase isoenzyme pro les between these speciesComparisons of our results with the published RFLPpro le of H oryzicola (Subbotin et al 2000a) supportthe use of the enzymes BsuRI CfoI MvaI RsaI forseparation of these species from each other Comparisonof RFLP pro les obtained for the Iranian H elachistapopulation with that from Japan (Orui 1997) con rmstheir conspeci c status

Heterodera mothi was rst recorded in Iran by Ta-lachian et al (1976) Plants of the genus Cyperus whichare hosts for this species are dominant weeds in Iranianrice elds and the probability of joint occurrence ofH mothi and H elachista in rice elds is therefore highThe ITS-RFLP of Bsh1236I and BsuRI separates thesespecies

108 Nematology

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Cyst nematodes from Iran

Heterodera sp 4 found on the roots of Phragmitessp was morphologically identi ed as a member of theH cardiolata Kirjanova amp Ivanova 1969 complex in-cluding H cardiolata H graminis Stynes 1971 H cyno-dontis parasitising Cynodon dactylon and H phragmi-tidis Kazachenko 1986 parasitising Phragmites australis(Cav) Trin ex Steud Further morphometric analysis ofthe J2 and cysts revealed differences between the Iranianpopulation and these species (Tanha Maa unpubl) Thesequence of Heterodera sp 4 was different from to that ofH cynodontis Based on the molecular and morphometricresults this species can be considered as an undescribedspecies

Heterodera sp 5 morphologically identi ed as an un-known member of the Goettingianagroup shows geneticsimilarity with species from this group Detailed morpho-logical and morphometric study of the J2 and cysts re-vealed differences between this species and H goettin-giana H cruciferae H carotae and H urticae (TanhaMaa unpubl)Molecular analysis also suggests that thispopulationbelongs to an undescribed species

Another member of the Goettingiana group Hetero-dera sp 6 collected from soil around the root of kiwiand peach trees morphologically and morphometricallyresembles H cruciferae However RFLP generated byBsh1236I CfoI and RsaI distinguished these populationsfrom a Dutch populationof H cruciferae (Subbotin et al2000a) Heterodera sp 7 found in soil samples from ap-ple orchards was morphometrically and morphologicallysimilar to H urticae but Bsh1236I and RsaI digestion re-sulted in different patterns for these nematodes

Phylogenetic relationships within cyst-forming nema-todes based on the ITS sequences together with the in- uence of alignment procedures and methods of phy-logenetic inference were recently analysed (Subbotin etal 2001) The phylogenetic trees obtained in the presentstudy are in accordance with these published results Thefact that H turcomanica clusters with high bootstrapsupport with the Humuli group and does not belong tothe Avenae group is a novel conclusion from our studyIt supports the opinion of Sturhan (pers comm) thatthis species is clearly not related to the H avenae andH latipons species complex Present data do not supportthe validity of the genus Ephippiodera proposed by Sha-galina and Krall (1981) for the species H turcomanicaand H latipons and justify the synonymisation of Ephip-piodera with Heterodera (Luc et al 1988 Sturhan ampWouts 1995)

Nobbs et al (1992) suggested that an additional speciesgroup was appropriate for H cyperi Golden Rau amp Cobb1962 H elachista H oryzicola and H mothi speciescharacterised by the presence of bullae and a weak orabsent underbridge Subbotin et al (2001) based onphylogenetic analyses of sequences from the ITS regionof rDNA and morphological characters of cyst and J2erected a new group the Cyperi group for H cyperiand H oryzicola Clustering in our trees of the newlysequenced H elachista and H mothi with H oryzicolaand H cyperi therefore fully justify the erection of theCyperi group

Acknowledgements

The rst author is grateful for the awarding of ascholarship from the Agricultural Research Educationand Extension Organisation and Plant Pests and DiseasesResearch Institute Tehran Iran SA Subbotin gratefullyacknowledges nancial support from a NATO ResearchFellowship

References

BAROOTI S amp LOOF PAA (1990) The cereal cyst nema-tode Heterodera avenae occurring on wheat in Marvast areaof Iran Abstracts of the Second International NematologyCongress August 1990 Wageningen The Netherlands 25376 [Abstr]

CAVENESS EE amp JENSEN HJ (1955) Modi cation ofthe centrifugal otation technique for the isolation andconcentration of nematodes and their eggs from soil andplant tissue Proceeding of the Helminthological Society ofWashington 22 87-89

CLAPP JP VAN DER STOEL CD amp VAN DER PUTTEN WH (2000) Rapid identi cation of cyst (Heterodera sppGlobodera spp) and root-knot (Meloidogyne spp) nematodeson the basis of ITS2 sequence variation detected by PCR-single-strand conformational polymorphism (PCR-SSCP) incultures and eld samples Molecular Ecology 9 1223-1232

DAMADZADEH M amp ANSARIPOUR B (2001) Identi cationand distribution of Heterodera lipjevi in the Esfahan area ofIran Russian Journal of Nematology 9 57-58

DE GRISSE A (1969) Redescription ou modi cations dequelques techniques utiliseacutees dans lrsquoeacutetude des neacutematodesphytoparasitaires Mededelingen Rijksfaculteit der Land-bouwwetenschappen Gent 34 351-369

DUNN RA (1969) Extraction of cysts of Heterodera speciesfrom soils by centrifugationin high density solutions Journalof Nematology 1 7

Vol 5(1) 2003 109

Z Tanha Maa et al

EROSHENKO AS SUBBOTIN SA amp KAZACHENKO IP(2001) Heterodera vallicola sp n (Tylenchida Heteroderi-dae) from elm trees Ulmus japonica (Rehd) Sarg in the Pri-morsky territory the Russian Far East with rDNA identi ca-tion of closely related species Russian Journal of Nemato-logy 9 9-17

ESMAILPOUR MH amp SCHAumlFFER R (1970) Sugar beet ne-matode Heterodera schachtii in Iran Entomologie et Phy-topathologie Appliqueacutees 29 6-7

FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM MA(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151

FERRIS VR SUBBOTIN SA IREHOLM A SPIEGEL Y FAGHIHI J amp FERRIS JM (1999) Ribosomal DNAsequence analysis of Heterodera lipjevi and H latiponsisolates from Russia and comparisons with other nematodeisolates Russian Journal of Nematology 7 121-125

GAumlBLER C STURHAN D SUBBOTIN SA amp RUMPEN-HORST HJ (2000) Heterodera pratensis sp n a new cystnematode of the H avenae complex (Nematoda Heteroderi-dae) Russian Journal of Nematology 8 115-126

GOLDEN AM (1986) Morphology and identi cation of cystnematodes In Lamberti F amp Taylor CE (Eds) Cystnematodes New York amp London Plenum Press pp 23-45

HOJAT-JALALI AA (1991) An investigation on cerealcyst nematode (CNN) (Heterodera avenae) in BakhtaranProvince Iranian Journal of Plant Pathology 27 99

JOYCE SA REID A DRIVER F amp CURRAN J (1994)Application of polymerase chain reaction (PCR) methods toidenti cation of entomopathogenic nematodes In BurnellAM Ehlers R-U amp Masson JP (Eds) Cost 812 Biotech-nology Genetics of entomopathogenic nematode-bacteriumcomplexes Proceedings of Symposium amp Workshop StPatrick s College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187

LUC M MAGGENTI AR amp FORTUNER R (1988) A reap-praisal of Tylenchina (Nemata) 9 The family HeteroderidaeFilipjev amp Schuurmans Stekhoven1941 Revue de Neacutematolo-gie 11 159-176

MEHDIKHANI MOGHADAM E (1998) Identi cation of twospecies of Heterodera in sugar beet elds in Mashhad regionProceedings of 13th Iranian Plant Protection CongressKaraj 139 [Abstr]

MEHDIKHANI MOGHADAM E amp KHEIRI A (1995) Someplant parasitic nematode fauna of sugar beet elds in Mash-had region Iranian Journal of Plant Pathology 31 24-26

MEHDIKHANI MOGHADAM E KHEIRI A amp OKHOVAT M(1996) Morphological and morphometrical study of threeendoparasitic nematodes of sugar beet in Mashhad regionIranian Journal of Plant Pathology 32 1-2

MULVEY RH (1972) Identi cation of Heterodera cysts byterminal and cone top structures Canadian Journal of Zool-ogy 50 1-59

MULVEY RH amp GOLDEN AM (1983) An illustrated keyto the cyst-forming genera and species of Heteroderidaein the western hemisphere with species morphometrics anddistribution Journal of Nematology 5 1-59

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110 Nematology

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