Nematology, 2000, Vol. 2(2), 153-164 Identi cation of cyst forming nematodes of the genus Heterodera (Nematoda: Heteroderidae) based on the ribosomal DNA-RFLP Sergei A. SUBBOTIN 1, * , Lieven WAEYENBERGE 2 and Maurice MOENS 2 1 Institute of Parasitology of Russian Academy of Sciences, Leninskii prospect 33, Moscow, 117071, Russia; 2 Agricultural Research Centre, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium Accepted for publication:31 August 1999 Summary – Ampli ed ITS region products of rDNA from 25 valid species and one unidenti ed species from the genus Heterodera and from Meloidodera alni were digested by 26 restriction enzymes. A combination of seven enzymes clearly separated the agriculturally most important species from each other and from their sibling species. Species speci c digestion pro les of ITS regions and a table with approximate sizes of digested fragments for several identi cation enzymes are given. Heterogeneity of ITS regions was revealed for some cyst forming nematode species. Résumé – Identi cation de nématodes à kystes du genre Heterodera (Nematoda: Heteroderidae) basée sur les RFLP du DNA ribosomal – Des fragments ampli és de la région de l’ITS du rDNA de 25 espèces valides et d’une espèce non identi ée du genre Heterodera et de Meloidodera alni ont été soumis à une digestion par 26 enzymes de restriction. La combinaison de sept enzymes a permis une séparation nette des espèces les plus importantes en agriculture, tant les unes par rapport aux autres que par rapport aux espèces jumelles. Sont donnés les pro ls spéci ques de digestion des régions de l’ITS et un tableau regroupant les tailles approximatives des fragments digérés pour plusieurs enzymes d’identi cation. L’hétérogénéité des régions de l’ITS a été révélée chez quelques espèces de nématodes à kyste. Keywords – heterogeneity,ITS regions, Meloidodera alni. The genus Heterodera belongsto the family Heteroderi- dae and contains 62 species (Wouts & Baldwin, 1998). Some of them, H. avenae , H. cajani , H. cruciferae , H. li- pjevi , H. glycines , H. goettingiana , H. medicaginis , H. or- yzicola, and H. schachtii , are important agricultural pests for European and global agriculture(Baldwin & Mundo- Ocampo, 1991; Evans & Rowe, 1998). Most of the Hete- rodera species are classi ed into so-called ‘species com- plexes’ each grouping several sibling species. Identi ca- tion of these cyst forming nematodes based on differences in morphological and morphometric characters requires a lot of skill, is time consuming and often inconclusive for individuals.However, as most of the modern plant protec- tion measures are species speci c, accurate, fast and reli- able identi cation of nematode populationsat the species and subspecies level becomes more and more important. Developed during the last decade, DNA-based diag- nostics provide an attractive solution to problems asso- ciated with traditional identi cation methods. Compara- * Corresponding author, e-mail: [email protected]Current address: Agricultural Research Centre, Burg. Van Gansberghelaan 96, 9820 Merelbeke, Belgium. tive analyses of coding and non-coding regions of ribo- somal DNA (rDNA) became a popular tool for species and subspecies identi cation of plant-parasiticnematodes from many genera (Vrain et al., 1992; Wendt et al., 1993; Zijlstra et al., 1995). RFLP analyses of ribosomal inter- nal transcribed spacer regions (ITS) were useful for the identi cation of Globodera species parasitic to solana- ceous plants (Thiéry & Mugniéry, 1996). At least three restriction site differences were identi ed to discriminate Globodera pallida and G. rostochiensis , the two potato cyst nematode species (Fleming & Powers, 1998; Sub- botin et al. , 1999a). Ferris et al. (1993, 1994), on the ba- sis of sequence data from the ITS region, showed that the genus Heterodera exhibited a rather wide range of ge- netic distances. However, within the H. schachtii group, the clover cyst nematode, H. trifolii , and the soybean cyst nematode, H. glycines , differed by only a few nucleotide substitutions. RFLP analysis of rDNA was also used for the identi cation of H. cruciferae , H. glycines , H. trifolii , c ® Koninklijke Brill NV, Leiden, 2000 153
Transcript
Nematology 2000 Vol 2(2) 153-164
Identi cation of cyst forming nematodes of the genus Heterodera(Nematoda Heteroderidae) based on the ribosomal DNA-RFLP
Sergei A SUBBOTIN 1 Lieven WAEYENBERGE 2 and Maurice MOENS 2
1 Institute of Parasitology of Russian Academy of Sciences Leninskii prospect 33 Moscow 117071 Russia2 Agricultural Research Centre Burg Van Gansberghelaan 96 9820 Merelbeke Belgium
Accepted for publication31 August 1999
Summary ndash Ampli ed ITS region products of rDNA from 25 valid species and one unidenti ed species from the genus Heterodera andfrom Meloidodera alni were digested by 26 restriction enzymes A combination of seven enzymes clearly separated the agriculturallymost important species from each other and from their sibling species Species speci c digestion pro les of ITS regions and a tablewith approximate sizes of digested fragments for several identi cation enzymes are given Heterogeneity of ITS regions was revealedfor some cyst forming nematode species
Reacutesumeacute ndash Identi cation de neacutematodes agrave kystes du genre Heterodera (Nematoda Heteroderidae) baseacutee sur les RFLP du DNAribosomal ndash Des fragments ampli eacutes de la reacutegion de lrsquoITS du rDNA de 25 espegraveces valides et drsquoune espegravece non identi eacutee du genreHeterodera et de Meloidodera alni ont eacuteteacute soumis agrave une digestion par 26 enzymes de restriction La combinaison de sept enzymes apermis une seacuteparation nette des espegraveces les plus importantes en agriculture tant les unes par rapport aux autres que par rapport auxespegraveces jumelles Sont donneacutes les pro ls speacuteci ques de digestion des reacutegions de lrsquoITS et un tableau regroupant les tailles approximativesdes fragments digeacutereacutes pour plusieurs enzymes drsquoidenti cation Lrsquoheacuteteacuterogeacuteneacuteiteacute des reacutegions de lrsquoITS a eacuteteacute reacuteveacuteleacutee chez quelques espegravecesde neacutematodes agrave kyste
Keywords ndash heterogeneity ITS regions Meloidodera alni
The genusHeterodera belongs to the family Heteroderi-dae and contains 62 species (Wouts amp Baldwin 1998)Some of them H avenae H cajani H cruciferae H li-pjevi H glycines H goettingiana H medicaginis H or-yzicola and H schachtii are important agricultural pestsfor European and global agriculture (Baldwin amp Mundo-Ocampo 1991 Evans amp Rowe 1998) Most of the Hete-rodera species are classi ed into so-called lsquospecies com-plexesrsquo each grouping several sibling species Identi ca-tion of these cyst forming nematodes based on differencesin morphological and morphometric characters requires alot of skill is time consuming and often inconclusive forindividualsHowever as most of the modern plant protec-tion measures are species speci c accurate fast and reli-able identi cation of nematode populations at the speciesand subspecies level becomes more and more important
Developed during the last decade DNA-based diag-nostics provide an attractive solution to problems asso-ciated with traditional identi cation methods Compara-
Corresponding author e-mail ssubbotinclofgovbeCurrent address Agricultural Research Centre Burg Van Gansberghelaan96 9820 Merelbeke Belgium
tive analyses of coding and non-coding regions of ribo-somal DNA (rDNA) became a popular tool for speciesand subspecies identi cation of plant-parasitic nematodesfrom many genera (Vrain et al 1992 Wendt et al 1993Zijlstra et al 1995) RFLP analyses of ribosomal inter-nal transcribed spacer regions (ITS) were useful for theidenti cation of Globodera species parasitic to solana-ceous plants (Thieacutery amp Mugnieacutery 1996) At least threerestriction site differences were identi ed to discriminateGlobodera pallida and G rostochiensis the two potatocyst nematode species (Fleming amp Powers 1998 Sub-botin et al 1999a) Ferris et al (1993 1994) on the ba-sis of sequence data from the ITS region showed that thegenus Heterodera exhibited a rather wide range of ge-netic distances However within the H schachtii groupthe clover cyst nematode H trifolii and the soybean cystnematode H glycines differed by only a few nucleotidesubstitutions RFLP analysis of rDNA was also used forthe identi cation of H cruciferae H glycines H trifolii
creg Koninklijke Brill NV Leiden 2000 153
SA Subbotin et al
H schachtii H goettingianaand H zeae (Szalanski et al1997) With the same technique Subbotin et al (1997)differentiated species from the H humuli group whereasBekal et al (1997) and Subbotin et al (1999b) separatedspecies within the H avenae group and Orui (1997) andFleming et al (1998) some other cyst nematodes
In the present paper we report on the use of rDNARFLP in distinguishing Heterodera species on the cre-ation of species-speci c digestion pro les and on ourstudy of the variation of RFLP patterns between popula-tions of some species
Materials and methods
NEMATODE ISOLATES
The present study comprised 25 valid Heteroderaspecies (Table 1) all identi ed by their morphology andmorphometrics One Heterodera population collectedfrom Cynodon dactylon and similar to H cardiolata wasalso included To determine the degree of intraspeci cvariation some of the species were represented by sev-eral populations The species were categorised into fourgroups H avenae H schachtii H humuli and H goettin-giana according to the criteria and classi cations of Math-ews (1971) Baldwin and Mundo-Ocampo (1991) andWouts and Sturhan (1995) The H avenae group was fur-ther split into two subgroups H avenae sensu stricto andH latipons The H schachtii group contained a subgroupH schachtii s str composed of H schachtii H trifoliiH glycines H ciceri and H medicaginis Previously ob-tained RFLP data of populationsfrom the H humuli group(Subbotin et al 1997) and the H avenae group(Subbotinet al 1999b) were included in the analysis Two popula-tions of Meloidodera alni were used for comparison
SAMPLE PREPARATION
For each population one to four cysts were transferredinto 10 m l of double distilled water in an Eppendorftube and crushed with a microhomogenisator Eight m l ofnematode lysis buffer (125 mM KCl 25 mM Tris-Cl pH83 375 mM MgCl2 25 mM DTT 1125 Tween 200025 gelatine) and 2 m l of proteinase K (600 mgml)were added The tubes were incubated at 65degC (1 h) and95degC (10 min) consecutively
PCR REACTION
After centrifugation (1 min 16 000 g) 10 m l of theDNA suspension was added to the PCR reaction mix-ture containing 10 m l 10X Taq incubation buffer 20 m l5X Q-solution 200 mM of each dNTP (Taq PCR CoreKit Qiagen Germany) 15 mM of each primer (synthe-sised by Life Technologies Merelbeke Belgium) 08UTaq Polymerase (Taq PCR Core Kit Qiagen Germany)and double distilled water to a nal volume of 100 m lPrimers AB 28 (5 cent -ATATGCTTAAGTTCAGCGGGT-3 cent )and TW 81 (5 cent -GTT-TCCGTAGGTGAACCTGC-3 cent ) asdescribed by Joyce et al (1994) were used in the PCRreaction The DNA-ampli cation pro le carried out in aGeneE (New Brunswick Scienti c Wezembeek-OppemBelgium) DNA thermal cycler consisted of 4 min 94degC35 cycles of 1 min 94degC 15 min 55deg C and 2 min 72degCfollowed by a nal elongation step of 10 min 72degC AfterDNA ampli cation 5 m l product was run on a 1 agarosegel The remainder was stored at shy 20degC
RFLP
Five to 7 m l of each PCR product was digested withone of the following 26 restriction enzymes AluI AvaIBamHI BglI BsiZI BsuRI Bsh1236I Bsp143I CfoIDdeI EcoRI HpaII HindIII HinfI KpnI MvaI PstIPvuII RsaI SalI SfuI SspI ScrFI TaqI Tru9I and XbaIin the buffer stipulated by the manufacturer The digestedDNA was loaded on a 15 agarose gel separated byelectrophoresis (100V 25 h) stained with ethidium bro-mide visualised on a 2011 Macrovue UV transillumina-tor and photographed with a Polaroid MP4+ InstantCamera System Procedures for obtaining PCR ampli edproducts and endonuclease digestion of these productswere repeated several times to verify the results Poorlyvisible fragments less than 100 bp and some weak addi-tional restriction fragments have not been taken into ac-count
Results
Ampli cation of the rDNA-ITS regionswith the presentprimers was successful for all species Most species yield-ed a single fragment of approximately1060 bp Howeverthe PCR ampli ed product of H cyperi was near 1100 bpand that of H oryzicola near 1010 bp No PCR productswere obtained in the negative control lacking DNA tem-plate
154 Nematology
Identication of Heterodera species
Table 1 Nematode species and populations used in this study
Species Group or Location Hosts Source Code Tested enzymessubgroup
H avenae1) avenaes st Rinkam Bavaria Cereals D Sturhan Germany H1 All(type A) Germany
H avenae avenaes st Unknown India Cereals J Rowe UK H2 All(type B)
H arenaria avenaes st Lincolnshire England Ammophila J Rowe UK H3 Allarenaria
H lipjevi2) avenaes st Vad Russia Avena sativa L Nasonova Russia H4 AllH aucklandica avenaes st One Tree Hill Auckland Microlaena W Wouts New Zealand H5 All
New Zealand stipoidesH iri avenaes st Forfar Scotland UK Grasses SA Subbotin Russia H6a AllH iri avenaes st Near Belgium Grasses SA Subbotin Russia H6b AluI Bsp143I BsiZI
CfoI ScrFI Tru9IH latipons latipons Rostov region Russia Elytrigia repens SA Subbotin Russia H7 AllH hordecalis latipons Montrose Scotland UK Grasses SA Subbotin Russia H8a AllH hordecalis latipons Unknown Sweden Cereals A Ireholm Sweden H8b AluI BsiZI Bsp143I
BsuRI CfoI DdeIHpaII MvaI PvuII
RsaI ScrFI SspIH schachtii schachtii s st Unknown Germany Beta vulgaris D Sturhan Germany H9a AllH schachtii schachtii s st Hem The Netherlands Beta vulgaris B Schoemaker H9b HpaII MvaI
The Netherlands RsaI PvuIIH schachtii schachtii s st Unknown Belgium Beta vulgaris M Moens Belgium H9c HpaII MvaI
RsaI PvuIIH trifolii schachtii s st Unknown UK Trifolium sp J Rowe UK H10a AllH trifolii schachtii s st Brussegem Belgium Trifolium sp SA Subbotin Russia H10b AluI CfoI HpaII
MvaI RsaI ScrFIH medicaginis schachtii s st Stavropol region Russia Medicago sativa SA Subbotin Russia H11 AllH ciceri schachtii s st Unknown Italy Cicer sp N Vovlas Italy H12 AllH salixophila schachtii Kherson Ukraine Salix album SA Subbotin Russia H13a AllH salixophila schachtii Nieuwpoort Belgium Salix sp SA Subbotin Russia H13b AluI Bsp143I BsuRI
CfoI HinfI MvaIPstI RsaI TaqI
H oryzicola schachtii Kerala India Oryza sativa J Rowe UK H14 AllH glycines schachtii s st Arkansas USA Glycinemax R Robbins USA H15 AllH cajani schachtii Unknown India Cajanuscajan J Rowe UK H16 AllH humuli3) humuli Poperinge Belgium Humuluslupulus SA Subbotin Russia H17 AllH riparia4) humuli Moscow region Russia Urtica dioica SA Subbotin Russia H18a AllH riparia humuli St Albans UK Urtica sp SA Subbotin Russia H18b AluI CfoI PstI
RsaI Tru9IH ci humuli Sukhumi Georgia Ficuscarica SA Subbotin Russia H19 AllH litoralis humuli Glen Innes Auckland Sarcocornia W Wouts New Zealand H20 All
New Zealand uinque oraH carotae goettingiana Sion Wallis Switzerland unknown J Grunder Switzerland H21a AllH carotae goettingiana Creacuteances France Daucussp M Bossis France H21b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH cruciferae goettingiana Brielle The Netherlands unknown B Schoemaker H22 All
The NetherlandsHeterodera sp goettingiana Kherson Ukraine Cynodondactylon SA Subbotin Russia H23 AllH cyperi goettingiana Unknown Spain Cyperus sp M Romero Spain H24 AllH goettingiana goettingiana Unknown Germany Pisumsp J Rowe UK H25a AllH goettingiana goettingiana Vieille-Eglise France Pisumsativum M Moens Belgium H25b AluI Bsh1236I
CfoI HpaII RsaIH urticae goettingiana Luxembourg province Urtica sp SA Subbotin Russia H26a All
BelgiumH urticae goettingiana Diksmuide Belgium Urtica sp SA Subbotin Russia H26b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH urticae goettingiana Near Belgium Urtica sp SA Subbotin Russia H26c AluI Bsh1236I CfoI
HpaII RsaIMeloidodera alni Luxembourg province Alnus sp SA Subbotin Russia M27a All
BelgiumMeloidodera alni Moscow region Russia Alnus incana VN Chizhov Russia M27b AluI Bsh1236I Bsp143I
PCR product were digested by AluI Bsh1236I Bsp143I BsuRI CfoI HindIII HinfI HpaII MvaI PstI RsaI and TaqI from 1) eight additional populations of H avenae2) seven of H lipjevi (Subbotin et al 1999b) and 3) two of H humuli 4) four of H riparia (Subbotin et al 1997)
Vol 2(2) 2000 155
SA Subbotin et al
Digestion with 23 out of the 26 enzymes gave RFLPsfor all species studied BamHI HindIII and KpnI didnot restrict any of the ampli ed products No singleenzyme could distinguish all cyst forming species inthis study although some yielded much more taxonomicinformation than others (Table 2) For example CfoI(Fig 1G) yielded 16 polymorphic patterns distinguishing12 out of the 26 species AluI (Fig 1A) BsuRI (Fig 1F)Bsh1236I (Fig 1C) and ScrFI (Fig 2F) each produced15 polymorphic patterns and distinguished nine ten 11and nine species respectively The combination of thepatterns obtained by seven individual enzymes alloweddifferentiation of most species under study (Table 3) Insome cases the sum of restricted fragments length was lessthan 1060 bp This could be due to production of severalfragments with similar sizes orand of fragments smallerthat 100 bp which were poorly visible on the agarosegels Sometimes the sum of restricted fragment lengthswas more than the length of the unrestricted ampli edproduct Repeated digestion with an extended digestionperiod suggestedheterogeneityof ITS regionswas presentin the genome of these species
RFLP of ITS regions allowed clear differentiation ofmost agriculturally important cyst forming nematode spe-cies from each other and from their sibling species Somerestriction enzymes produced a RFLP pattern speci c fora species group For example the patterns obtained af-ter restriction with AluI (Fig 1A) Bsh1236I (Fig 1C)BsuRI (Fig 1F) DdeI (Fig 1H) HinfI (data not shown)and EcoRI (Fig 2A) distinguished the H schachtii s strgroup from the other groups whereas EcoRI restrictedonly ITS regions of species from this group BglI (datanot shown) BsiZI (Fig 1D) Bsp143I (Fig 1E) BsuRI(Fig 1F) and PstI (Fig 2D) separated the H goettin-giana group species having juveniles with four incisures(H carotae H cruciferae H goettingiana and H ur-ticae) from other species Some enzymes proved to be oflittle use for identi cation of larger numbers of the speciesbut were extremely adequate for individual species SspI(data not shown) for example only restricted PCR prod-ucts from H latipons and M alni whereas the ITS fromHeterodera sp was only cut by XbaI (data not shown)BsiZI (Fig 1D) and DdeI (Fig 1H) restricted the ITS re-gion of all species except H litoralis
As we observed earlier (Subbotin et al 1997) Eu-ropean populations of H avenae (type A) were easilyseparated from other species with AluI (Fig 1A) whichdigested the ITS regions of all studied nematodes ex-cept those of H arenaria No enzymes distinguished
European populations of H avenae from H arenariaH lipjevi was easily distinguished from all species byPstI (Fig 2D) BsuRI (Fig 1F) and TaqI (Fig 2H)which produced unique RFLP patterns for this speciesH aucklandica was separated by CfoI (Fig 1G) fromother species Bsh1236I (Fig 1C) CfoI (Fig 1G) andScrFI (Fig 2F) produceduniquepatterns for H iri H lati-pons was separated from the others by AluI (Fig 1A)Bsh1236I(Fig 1C) BsuRI (Fig 1F) CfoI (Fig 1G) DdeI(Fig 1H) PvuII RsaI (Fig 2E) and SspI H hordecaliswas distinguishedby Bsh1236I(Fig 1C) BsuRI (Fig 1F)MvaI (Fig 2C) and ScrFI (Fig 2F)
AluI AvaI CfoI HpaII MvaI RsaI and ScrFI sepa-rated the closely related and morphologically poorly dis-tinguished species from the H schachtii s str group(H schachtii H glycines H trifolii H medicaginis andH ciceri) from each other and all other species MvaI(Fig 2C) PvuII RsaI (Figs 2E 3) and ScrFI (Fig 2F)produced a speci c pattern for H schachtii which sep-arated this species from others Restrictions with MvaIRsaI HpaII or PvuII did not show intraspeci c variationin restriction patterns between the three H schachtii pop-ulations under study (Fig 3) Totalling the fragment sizesproduced by MvaI (Figs 2C 3) RsaI (Figs 2E 3) PvuII(Fig 3) or ScrFI (Fig 2F) yielded a sum of more than1060 bp indicatinga heterogeneityof ITS regions presentin the genome of H schachtii populations H glycinesanother member of the H schachtii s str group was dis-tinguished within this group by AvaI which produced apattern identical to that for H cajani (Fig 1B) H ca-jani was easily separated from others by many enzymesAluI (Fig 1A) Bsh1236I (Fig 1C) BsiZI (Fig 1D)BsuRI (Fig 1F) CfoI (Fig 1G) DdeI (Fig 1H) HinfIHpaII (Fig 2B) ScrFI (Fig 2F) TaqI (Fig 2H) andTru9I RFLP patterns produced by CfoI (Fig 1G) sep-arated H ciceri from others Heterogeneity of the ITSregion was observed for H ciceri with AluI (Fig 1A)CfoI (Fig 1G) RsaI (Fig 2E) Combinations of at leasttwo restriction enzymes separated other species from thisH schachtii s str group For example RsaI (Fig 2E) dis-tinguished H trifolii and H ciceri from other specieswhilst CfoI (Fig 1G) separated these two species RsaI(Fig 2E) demonstrated heterogeneity of ITS regions intwo populations of H trifolii and AluI in one populationof this species H medicaginis was distinguished fromother species by a combination of three enzymes HpaII(Fig 2B) MvaI (Fig 2C) and AvaI (Fig 1B Table 3)H salixophila belonging to the H schachtii group wasseparated from all species studied by Bsh1236I (Fig 1C)
156 Nematology
Identication of Heterodera species
Tab
le2
Num
ber
ofdi
ffer
entR
FL
Ppr
ole
s1 )yi
elde
dby
asi
ngle
enzy
me
dige
stio
nof
the
ITS
regi
ons
ofcy
stne
mat
odes
Spe
cies
Alu
IA
vaI
Bgl
IB
sh12
36I
Bsi
ZI
Bsp
143I
Bsu
RI
Cfo
ID
deI
Eco
RI
Hin
fIH
paII
Mva
IP
stI
Pvu
IIR
saI
Sal I
Scr F
ISf
u ISs
p ITa
q ITr
u 9I
Xba
I
Ha
vena
e(t
ype
A)
11
11
1a1
11
11
11
11
11
11
11
11
11
Ha
vena
e(t
ype
B)
21
11
11
11
11
11
11
12
11
11
11
1H
are
nari
a1
11
11
11
11
11
11
11
11
11
11
11
H
lipj
evi
21
11
11
21
11
21
12
12
11
11
22
1H
auc
klan
dica
21
11
11
12
11
11
11
12
11
11
31
1H
iri
21
12
22
13
11
22
21
12
12
11
32
1H
lat
ipon
s3
11
33
23
42
12
32
12
31
31
23
31
Hh
orde
cali
s4
11
43
14
11
12
33
11
11
41
14
11
Hs
chac
htii
52
15
41
55
32
34
41
1a4a
15a
11
31
1H
tri
foli
i5
5a2
15
41
55
32
32
51
34b
16
11
31
1H
med
icag
inis
52
15
41
55
32
34
51
34
15
11
31
1H
cic
eri
5a2
15
41
55a
32
32
51
34b
16
11
31
1H
sal
ixop
hila
23
16
51
66
41
45
11
15
71
32
15
41
Ho
ryzi
cola
61
17
63
77
51
55
63
46
17
31
65
1H
gly
cine
s5
41
54
15
53
23
45
13
41
51
13
11
Hc
ajan
i7
41
87
18
86
16
67
13
71
82
17
61
Hh
umul
i8
31
18
19
97
17
77
45
81
91
14
71
Hr
ipar
ia9
31
18
19
107
17
77
15
81
91
14
21
H
ci10
31
19
110
97
17
88
43
91
101
14
21
Hl
itor
alis
43
19
101
1111
81
77
94
310
111
11
42
1H
car
otae
111
210
114
1212
12a
91
72
75
111
112
11
48a
1H
cru
cife
rae
111
210
114
1212
91
72
75
111
112
11
48a
1H
eter
oder
asp
12
13
1112
513
1310
13
910
61
72
131
18
92
Hc
yper
i13
11
1213
614
1411
18
1011
76
123
144
19
101
Hg
oett
ingi
ana
141
213
114
1215
121
72
75
113
112
11
1011
1H
urt
icae
111
214
114
1212
91
73
75
111
11a
112
11
48
1M
eloi
dode
raal
ni15
11
1514
715
1613
19
1112
12a
11
141
151
34
31
1 )
num
bers
repr
esen
tid
enti
cal
rest
rict
ion
patt
erns
pa
tter
nsw
ith
aor
bha
vead
diti
onal
frag
men
ts
patt
erns
divi
ded
by
diff
ered
betw
een
popu
lati
ons
ofa
sing
lene
mat
ode
spec
ies
Vol 2(2) 2000 157
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
H schachtii H goettingianaand H zeae (Szalanski et al1997) With the same technique Subbotin et al (1997)differentiated species from the H humuli group whereasBekal et al (1997) and Subbotin et al (1999b) separatedspecies within the H avenae group and Orui (1997) andFleming et al (1998) some other cyst nematodes
In the present paper we report on the use of rDNARFLP in distinguishing Heterodera species on the cre-ation of species-speci c digestion pro les and on ourstudy of the variation of RFLP patterns between popula-tions of some species
Materials and methods
NEMATODE ISOLATES
The present study comprised 25 valid Heteroderaspecies (Table 1) all identi ed by their morphology andmorphometrics One Heterodera population collectedfrom Cynodon dactylon and similar to H cardiolata wasalso included To determine the degree of intraspeci cvariation some of the species were represented by sev-eral populations The species were categorised into fourgroups H avenae H schachtii H humuli and H goettin-giana according to the criteria and classi cations of Math-ews (1971) Baldwin and Mundo-Ocampo (1991) andWouts and Sturhan (1995) The H avenae group was fur-ther split into two subgroups H avenae sensu stricto andH latipons The H schachtii group contained a subgroupH schachtii s str composed of H schachtii H trifoliiH glycines H ciceri and H medicaginis Previously ob-tained RFLP data of populationsfrom the H humuli group(Subbotin et al 1997) and the H avenae group(Subbotinet al 1999b) were included in the analysis Two popula-tions of Meloidodera alni were used for comparison
SAMPLE PREPARATION
For each population one to four cysts were transferredinto 10 m l of double distilled water in an Eppendorftube and crushed with a microhomogenisator Eight m l ofnematode lysis buffer (125 mM KCl 25 mM Tris-Cl pH83 375 mM MgCl2 25 mM DTT 1125 Tween 200025 gelatine) and 2 m l of proteinase K (600 mgml)were added The tubes were incubated at 65degC (1 h) and95degC (10 min) consecutively
PCR REACTION
After centrifugation (1 min 16 000 g) 10 m l of theDNA suspension was added to the PCR reaction mix-ture containing 10 m l 10X Taq incubation buffer 20 m l5X Q-solution 200 mM of each dNTP (Taq PCR CoreKit Qiagen Germany) 15 mM of each primer (synthe-sised by Life Technologies Merelbeke Belgium) 08UTaq Polymerase (Taq PCR Core Kit Qiagen Germany)and double distilled water to a nal volume of 100 m lPrimers AB 28 (5 cent -ATATGCTTAAGTTCAGCGGGT-3 cent )and TW 81 (5 cent -GTT-TCCGTAGGTGAACCTGC-3 cent ) asdescribed by Joyce et al (1994) were used in the PCRreaction The DNA-ampli cation pro le carried out in aGeneE (New Brunswick Scienti c Wezembeek-OppemBelgium) DNA thermal cycler consisted of 4 min 94degC35 cycles of 1 min 94degC 15 min 55deg C and 2 min 72degCfollowed by a nal elongation step of 10 min 72degC AfterDNA ampli cation 5 m l product was run on a 1 agarosegel The remainder was stored at shy 20degC
RFLP
Five to 7 m l of each PCR product was digested withone of the following 26 restriction enzymes AluI AvaIBamHI BglI BsiZI BsuRI Bsh1236I Bsp143I CfoIDdeI EcoRI HpaII HindIII HinfI KpnI MvaI PstIPvuII RsaI SalI SfuI SspI ScrFI TaqI Tru9I and XbaIin the buffer stipulated by the manufacturer The digestedDNA was loaded on a 15 agarose gel separated byelectrophoresis (100V 25 h) stained with ethidium bro-mide visualised on a 2011 Macrovue UV transillumina-tor and photographed with a Polaroid MP4+ InstantCamera System Procedures for obtaining PCR ampli edproducts and endonuclease digestion of these productswere repeated several times to verify the results Poorlyvisible fragments less than 100 bp and some weak addi-tional restriction fragments have not been taken into ac-count
Results
Ampli cation of the rDNA-ITS regionswith the presentprimers was successful for all species Most species yield-ed a single fragment of approximately1060 bp Howeverthe PCR ampli ed product of H cyperi was near 1100 bpand that of H oryzicola near 1010 bp No PCR productswere obtained in the negative control lacking DNA tem-plate
154 Nematology
Identication of Heterodera species
Table 1 Nematode species and populations used in this study
Species Group or Location Hosts Source Code Tested enzymessubgroup
H avenae1) avenaes st Rinkam Bavaria Cereals D Sturhan Germany H1 All(type A) Germany
H avenae avenaes st Unknown India Cereals J Rowe UK H2 All(type B)
H arenaria avenaes st Lincolnshire England Ammophila J Rowe UK H3 Allarenaria
H lipjevi2) avenaes st Vad Russia Avena sativa L Nasonova Russia H4 AllH aucklandica avenaes st One Tree Hill Auckland Microlaena W Wouts New Zealand H5 All
New Zealand stipoidesH iri avenaes st Forfar Scotland UK Grasses SA Subbotin Russia H6a AllH iri avenaes st Near Belgium Grasses SA Subbotin Russia H6b AluI Bsp143I BsiZI
CfoI ScrFI Tru9IH latipons latipons Rostov region Russia Elytrigia repens SA Subbotin Russia H7 AllH hordecalis latipons Montrose Scotland UK Grasses SA Subbotin Russia H8a AllH hordecalis latipons Unknown Sweden Cereals A Ireholm Sweden H8b AluI BsiZI Bsp143I
BsuRI CfoI DdeIHpaII MvaI PvuII
RsaI ScrFI SspIH schachtii schachtii s st Unknown Germany Beta vulgaris D Sturhan Germany H9a AllH schachtii schachtii s st Hem The Netherlands Beta vulgaris B Schoemaker H9b HpaII MvaI
The Netherlands RsaI PvuIIH schachtii schachtii s st Unknown Belgium Beta vulgaris M Moens Belgium H9c HpaII MvaI
RsaI PvuIIH trifolii schachtii s st Unknown UK Trifolium sp J Rowe UK H10a AllH trifolii schachtii s st Brussegem Belgium Trifolium sp SA Subbotin Russia H10b AluI CfoI HpaII
MvaI RsaI ScrFIH medicaginis schachtii s st Stavropol region Russia Medicago sativa SA Subbotin Russia H11 AllH ciceri schachtii s st Unknown Italy Cicer sp N Vovlas Italy H12 AllH salixophila schachtii Kherson Ukraine Salix album SA Subbotin Russia H13a AllH salixophila schachtii Nieuwpoort Belgium Salix sp SA Subbotin Russia H13b AluI Bsp143I BsuRI
CfoI HinfI MvaIPstI RsaI TaqI
H oryzicola schachtii Kerala India Oryza sativa J Rowe UK H14 AllH glycines schachtii s st Arkansas USA Glycinemax R Robbins USA H15 AllH cajani schachtii Unknown India Cajanuscajan J Rowe UK H16 AllH humuli3) humuli Poperinge Belgium Humuluslupulus SA Subbotin Russia H17 AllH riparia4) humuli Moscow region Russia Urtica dioica SA Subbotin Russia H18a AllH riparia humuli St Albans UK Urtica sp SA Subbotin Russia H18b AluI CfoI PstI
RsaI Tru9IH ci humuli Sukhumi Georgia Ficuscarica SA Subbotin Russia H19 AllH litoralis humuli Glen Innes Auckland Sarcocornia W Wouts New Zealand H20 All
New Zealand uinque oraH carotae goettingiana Sion Wallis Switzerland unknown J Grunder Switzerland H21a AllH carotae goettingiana Creacuteances France Daucussp M Bossis France H21b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH cruciferae goettingiana Brielle The Netherlands unknown B Schoemaker H22 All
The NetherlandsHeterodera sp goettingiana Kherson Ukraine Cynodondactylon SA Subbotin Russia H23 AllH cyperi goettingiana Unknown Spain Cyperus sp M Romero Spain H24 AllH goettingiana goettingiana Unknown Germany Pisumsp J Rowe UK H25a AllH goettingiana goettingiana Vieille-Eglise France Pisumsativum M Moens Belgium H25b AluI Bsh1236I
CfoI HpaII RsaIH urticae goettingiana Luxembourg province Urtica sp SA Subbotin Russia H26a All
BelgiumH urticae goettingiana Diksmuide Belgium Urtica sp SA Subbotin Russia H26b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH urticae goettingiana Near Belgium Urtica sp SA Subbotin Russia H26c AluI Bsh1236I CfoI
HpaII RsaIMeloidodera alni Luxembourg province Alnus sp SA Subbotin Russia M27a All
BelgiumMeloidodera alni Moscow region Russia Alnus incana VN Chizhov Russia M27b AluI Bsh1236I Bsp143I
PCR product were digested by AluI Bsh1236I Bsp143I BsuRI CfoI HindIII HinfI HpaII MvaI PstI RsaI and TaqI from 1) eight additional populations of H avenae2) seven of H lipjevi (Subbotin et al 1999b) and 3) two of H humuli 4) four of H riparia (Subbotin et al 1997)
Vol 2(2) 2000 155
SA Subbotin et al
Digestion with 23 out of the 26 enzymes gave RFLPsfor all species studied BamHI HindIII and KpnI didnot restrict any of the ampli ed products No singleenzyme could distinguish all cyst forming species inthis study although some yielded much more taxonomicinformation than others (Table 2) For example CfoI(Fig 1G) yielded 16 polymorphic patterns distinguishing12 out of the 26 species AluI (Fig 1A) BsuRI (Fig 1F)Bsh1236I (Fig 1C) and ScrFI (Fig 2F) each produced15 polymorphic patterns and distinguished nine ten 11and nine species respectively The combination of thepatterns obtained by seven individual enzymes alloweddifferentiation of most species under study (Table 3) Insome cases the sum of restricted fragments length was lessthan 1060 bp This could be due to production of severalfragments with similar sizes orand of fragments smallerthat 100 bp which were poorly visible on the agarosegels Sometimes the sum of restricted fragment lengthswas more than the length of the unrestricted ampli edproduct Repeated digestion with an extended digestionperiod suggestedheterogeneityof ITS regionswas presentin the genome of these species
RFLP of ITS regions allowed clear differentiation ofmost agriculturally important cyst forming nematode spe-cies from each other and from their sibling species Somerestriction enzymes produced a RFLP pattern speci c fora species group For example the patterns obtained af-ter restriction with AluI (Fig 1A) Bsh1236I (Fig 1C)BsuRI (Fig 1F) DdeI (Fig 1H) HinfI (data not shown)and EcoRI (Fig 2A) distinguished the H schachtii s strgroup from the other groups whereas EcoRI restrictedonly ITS regions of species from this group BglI (datanot shown) BsiZI (Fig 1D) Bsp143I (Fig 1E) BsuRI(Fig 1F) and PstI (Fig 2D) separated the H goettin-giana group species having juveniles with four incisures(H carotae H cruciferae H goettingiana and H ur-ticae) from other species Some enzymes proved to be oflittle use for identi cation of larger numbers of the speciesbut were extremely adequate for individual species SspI(data not shown) for example only restricted PCR prod-ucts from H latipons and M alni whereas the ITS fromHeterodera sp was only cut by XbaI (data not shown)BsiZI (Fig 1D) and DdeI (Fig 1H) restricted the ITS re-gion of all species except H litoralis
As we observed earlier (Subbotin et al 1997) Eu-ropean populations of H avenae (type A) were easilyseparated from other species with AluI (Fig 1A) whichdigested the ITS regions of all studied nematodes ex-cept those of H arenaria No enzymes distinguished
European populations of H avenae from H arenariaH lipjevi was easily distinguished from all species byPstI (Fig 2D) BsuRI (Fig 1F) and TaqI (Fig 2H)which produced unique RFLP patterns for this speciesH aucklandica was separated by CfoI (Fig 1G) fromother species Bsh1236I (Fig 1C) CfoI (Fig 1G) andScrFI (Fig 2F) produceduniquepatterns for H iri H lati-pons was separated from the others by AluI (Fig 1A)Bsh1236I(Fig 1C) BsuRI (Fig 1F) CfoI (Fig 1G) DdeI(Fig 1H) PvuII RsaI (Fig 2E) and SspI H hordecaliswas distinguishedby Bsh1236I(Fig 1C) BsuRI (Fig 1F)MvaI (Fig 2C) and ScrFI (Fig 2F)
AluI AvaI CfoI HpaII MvaI RsaI and ScrFI sepa-rated the closely related and morphologically poorly dis-tinguished species from the H schachtii s str group(H schachtii H glycines H trifolii H medicaginis andH ciceri) from each other and all other species MvaI(Fig 2C) PvuII RsaI (Figs 2E 3) and ScrFI (Fig 2F)produced a speci c pattern for H schachtii which sep-arated this species from others Restrictions with MvaIRsaI HpaII or PvuII did not show intraspeci c variationin restriction patterns between the three H schachtii pop-ulations under study (Fig 3) Totalling the fragment sizesproduced by MvaI (Figs 2C 3) RsaI (Figs 2E 3) PvuII(Fig 3) or ScrFI (Fig 2F) yielded a sum of more than1060 bp indicatinga heterogeneityof ITS regions presentin the genome of H schachtii populations H glycinesanother member of the H schachtii s str group was dis-tinguished within this group by AvaI which produced apattern identical to that for H cajani (Fig 1B) H ca-jani was easily separated from others by many enzymesAluI (Fig 1A) Bsh1236I (Fig 1C) BsiZI (Fig 1D)BsuRI (Fig 1F) CfoI (Fig 1G) DdeI (Fig 1H) HinfIHpaII (Fig 2B) ScrFI (Fig 2F) TaqI (Fig 2H) andTru9I RFLP patterns produced by CfoI (Fig 1G) sep-arated H ciceri from others Heterogeneity of the ITSregion was observed for H ciceri with AluI (Fig 1A)CfoI (Fig 1G) RsaI (Fig 2E) Combinations of at leasttwo restriction enzymes separated other species from thisH schachtii s str group For example RsaI (Fig 2E) dis-tinguished H trifolii and H ciceri from other specieswhilst CfoI (Fig 1G) separated these two species RsaI(Fig 2E) demonstrated heterogeneity of ITS regions intwo populations of H trifolii and AluI in one populationof this species H medicaginis was distinguished fromother species by a combination of three enzymes HpaII(Fig 2B) MvaI (Fig 2C) and AvaI (Fig 1B Table 3)H salixophila belonging to the H schachtii group wasseparated from all species studied by Bsh1236I (Fig 1C)
156 Nematology
Identication of Heterodera species
Tab
le2
Num
ber
ofdi
ffer
entR
FL
Ppr
ole
s1 )yi
elde
dby
asi
ngle
enzy
me
dige
stio
nof
the
ITS
regi
ons
ofcy
stne
mat
odes
Spe
cies
Alu
IA
vaI
Bgl
IB
sh12
36I
Bsi
ZI
Bsp
143I
Bsu
RI
Cfo
ID
deI
Eco
RI
Hin
fIH
paII
Mva
IP
stI
Pvu
IIR
saI
Sal I
Scr F
ISf
u ISs
p ITa
q ITr
u 9I
Xba
I
Ha
vena
e(t
ype
A)
11
11
1a1
11
11
11
11
11
11
11
11
11
Ha
vena
e(t
ype
B)
21
11
11
11
11
11
11
12
11
11
11
1H
are
nari
a1
11
11
11
11
11
11
11
11
11
11
11
H
lipj
evi
21
11
11
21
11
21
12
12
11
11
22
1H
auc
klan
dica
21
11
11
12
11
11
11
12
11
11
31
1H
iri
21
12
22
13
11
22
21
12
12
11
32
1H
lat
ipon
s3
11
33
23
42
12
32
12
31
31
23
31
Hh
orde
cali
s4
11
43
14
11
12
33
11
11
41
14
11
Hs
chac
htii
52
15
41
55
32
34
41
1a4a
15a
11
31
1H
tri
foli
i5
5a2
15
41
55
32
32
51
34b
16
11
31
1H
med
icag
inis
52
15
41
55
32
34
51
34
15
11
31
1H
cic
eri
5a2
15
41
55a
32
32
51
34b
16
11
31
1H
sal
ixop
hila
23
16
51
66
41
45
11
15
71
32
15
41
Ho
ryzi
cola
61
17
63
77
51
55
63
46
17
31
65
1H
gly
cine
s5
41
54
15
53
23
45
13
41
51
13
11
Hc
ajan
i7
41
87
18
86
16
67
13
71
82
17
61
Hh
umul
i8
31
18
19
97
17
77
45
81
91
14
71
Hr
ipar
ia9
31
18
19
107
17
77
15
81
91
14
21
H
ci10
31
19
110
97
17
88
43
91
101
14
21
Hl
itor
alis
43
19
101
1111
81
77
94
310
111
11
42
1H
car
otae
111
210
114
1212
12a
91
72
75
111
112
11
48a
1H
cru
cife
rae
111
210
114
1212
91
72
75
111
112
11
48a
1H
eter
oder
asp
12
13
1112
513
1310
13
910
61
72
131
18
92
Hc
yper
i13
11
1213
614
1411
18
1011
76
123
144
19
101
Hg
oett
ingi
ana
141
213
114
1215
121
72
75
113
112
11
1011
1H
urt
icae
111
214
114
1212
91
73
75
111
11a
112
11
48
1M
eloi
dode
raal
ni15
11
1514
715
1613
19
1112
12a
11
141
151
34
31
1 )
num
bers
repr
esen
tid
enti
cal
rest
rict
ion
patt
erns
pa
tter
nsw
ith
aor
bha
vead
diti
onal
frag
men
ts
patt
erns
divi
ded
by
diff
ered
betw
een
popu
lati
ons
ofa
sing
lene
mat
ode
spec
ies
Vol 2(2) 2000 157
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
Identication of Heterodera species
Table 1 Nematode species and populations used in this study
Species Group or Location Hosts Source Code Tested enzymessubgroup
H avenae1) avenaes st Rinkam Bavaria Cereals D Sturhan Germany H1 All(type A) Germany
H avenae avenaes st Unknown India Cereals J Rowe UK H2 All(type B)
H arenaria avenaes st Lincolnshire England Ammophila J Rowe UK H3 Allarenaria
H lipjevi2) avenaes st Vad Russia Avena sativa L Nasonova Russia H4 AllH aucklandica avenaes st One Tree Hill Auckland Microlaena W Wouts New Zealand H5 All
New Zealand stipoidesH iri avenaes st Forfar Scotland UK Grasses SA Subbotin Russia H6a AllH iri avenaes st Near Belgium Grasses SA Subbotin Russia H6b AluI Bsp143I BsiZI
CfoI ScrFI Tru9IH latipons latipons Rostov region Russia Elytrigia repens SA Subbotin Russia H7 AllH hordecalis latipons Montrose Scotland UK Grasses SA Subbotin Russia H8a AllH hordecalis latipons Unknown Sweden Cereals A Ireholm Sweden H8b AluI BsiZI Bsp143I
BsuRI CfoI DdeIHpaII MvaI PvuII
RsaI ScrFI SspIH schachtii schachtii s st Unknown Germany Beta vulgaris D Sturhan Germany H9a AllH schachtii schachtii s st Hem The Netherlands Beta vulgaris B Schoemaker H9b HpaII MvaI
The Netherlands RsaI PvuIIH schachtii schachtii s st Unknown Belgium Beta vulgaris M Moens Belgium H9c HpaII MvaI
RsaI PvuIIH trifolii schachtii s st Unknown UK Trifolium sp J Rowe UK H10a AllH trifolii schachtii s st Brussegem Belgium Trifolium sp SA Subbotin Russia H10b AluI CfoI HpaII
MvaI RsaI ScrFIH medicaginis schachtii s st Stavropol region Russia Medicago sativa SA Subbotin Russia H11 AllH ciceri schachtii s st Unknown Italy Cicer sp N Vovlas Italy H12 AllH salixophila schachtii Kherson Ukraine Salix album SA Subbotin Russia H13a AllH salixophila schachtii Nieuwpoort Belgium Salix sp SA Subbotin Russia H13b AluI Bsp143I BsuRI
CfoI HinfI MvaIPstI RsaI TaqI
H oryzicola schachtii Kerala India Oryza sativa J Rowe UK H14 AllH glycines schachtii s st Arkansas USA Glycinemax R Robbins USA H15 AllH cajani schachtii Unknown India Cajanuscajan J Rowe UK H16 AllH humuli3) humuli Poperinge Belgium Humuluslupulus SA Subbotin Russia H17 AllH riparia4) humuli Moscow region Russia Urtica dioica SA Subbotin Russia H18a AllH riparia humuli St Albans UK Urtica sp SA Subbotin Russia H18b AluI CfoI PstI
RsaI Tru9IH ci humuli Sukhumi Georgia Ficuscarica SA Subbotin Russia H19 AllH litoralis humuli Glen Innes Auckland Sarcocornia W Wouts New Zealand H20 All
New Zealand uinque oraH carotae goettingiana Sion Wallis Switzerland unknown J Grunder Switzerland H21a AllH carotae goettingiana Creacuteances France Daucussp M Bossis France H21b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH cruciferae goettingiana Brielle The Netherlands unknown B Schoemaker H22 All
The NetherlandsHeterodera sp goettingiana Kherson Ukraine Cynodondactylon SA Subbotin Russia H23 AllH cyperi goettingiana Unknown Spain Cyperus sp M Romero Spain H24 AllH goettingiana goettingiana Unknown Germany Pisumsp J Rowe UK H25a AllH goettingiana goettingiana Vieille-Eglise France Pisumsativum M Moens Belgium H25b AluI Bsh1236I
CfoI HpaII RsaIH urticae goettingiana Luxembourg province Urtica sp SA Subbotin Russia H26a All
BelgiumH urticae goettingiana Diksmuide Belgium Urtica sp SA Subbotin Russia H26b AluI Bsh1236I CfoI
HpaII RsaI Tru9IH urticae goettingiana Near Belgium Urtica sp SA Subbotin Russia H26c AluI Bsh1236I CfoI
HpaII RsaIMeloidodera alni Luxembourg province Alnus sp SA Subbotin Russia M27a All
BelgiumMeloidodera alni Moscow region Russia Alnus incana VN Chizhov Russia M27b AluI Bsh1236I Bsp143I
PCR product were digested by AluI Bsh1236I Bsp143I BsuRI CfoI HindIII HinfI HpaII MvaI PstI RsaI and TaqI from 1) eight additional populations of H avenae2) seven of H lipjevi (Subbotin et al 1999b) and 3) two of H humuli 4) four of H riparia (Subbotin et al 1997)
Vol 2(2) 2000 155
SA Subbotin et al
Digestion with 23 out of the 26 enzymes gave RFLPsfor all species studied BamHI HindIII and KpnI didnot restrict any of the ampli ed products No singleenzyme could distinguish all cyst forming species inthis study although some yielded much more taxonomicinformation than others (Table 2) For example CfoI(Fig 1G) yielded 16 polymorphic patterns distinguishing12 out of the 26 species AluI (Fig 1A) BsuRI (Fig 1F)Bsh1236I (Fig 1C) and ScrFI (Fig 2F) each produced15 polymorphic patterns and distinguished nine ten 11and nine species respectively The combination of thepatterns obtained by seven individual enzymes alloweddifferentiation of most species under study (Table 3) Insome cases the sum of restricted fragments length was lessthan 1060 bp This could be due to production of severalfragments with similar sizes orand of fragments smallerthat 100 bp which were poorly visible on the agarosegels Sometimes the sum of restricted fragment lengthswas more than the length of the unrestricted ampli edproduct Repeated digestion with an extended digestionperiod suggestedheterogeneityof ITS regionswas presentin the genome of these species
RFLP of ITS regions allowed clear differentiation ofmost agriculturally important cyst forming nematode spe-cies from each other and from their sibling species Somerestriction enzymes produced a RFLP pattern speci c fora species group For example the patterns obtained af-ter restriction with AluI (Fig 1A) Bsh1236I (Fig 1C)BsuRI (Fig 1F) DdeI (Fig 1H) HinfI (data not shown)and EcoRI (Fig 2A) distinguished the H schachtii s strgroup from the other groups whereas EcoRI restrictedonly ITS regions of species from this group BglI (datanot shown) BsiZI (Fig 1D) Bsp143I (Fig 1E) BsuRI(Fig 1F) and PstI (Fig 2D) separated the H goettin-giana group species having juveniles with four incisures(H carotae H cruciferae H goettingiana and H ur-ticae) from other species Some enzymes proved to be oflittle use for identi cation of larger numbers of the speciesbut were extremely adequate for individual species SspI(data not shown) for example only restricted PCR prod-ucts from H latipons and M alni whereas the ITS fromHeterodera sp was only cut by XbaI (data not shown)BsiZI (Fig 1D) and DdeI (Fig 1H) restricted the ITS re-gion of all species except H litoralis
As we observed earlier (Subbotin et al 1997) Eu-ropean populations of H avenae (type A) were easilyseparated from other species with AluI (Fig 1A) whichdigested the ITS regions of all studied nematodes ex-cept those of H arenaria No enzymes distinguished
European populations of H avenae from H arenariaH lipjevi was easily distinguished from all species byPstI (Fig 2D) BsuRI (Fig 1F) and TaqI (Fig 2H)which produced unique RFLP patterns for this speciesH aucklandica was separated by CfoI (Fig 1G) fromother species Bsh1236I (Fig 1C) CfoI (Fig 1G) andScrFI (Fig 2F) produceduniquepatterns for H iri H lati-pons was separated from the others by AluI (Fig 1A)Bsh1236I(Fig 1C) BsuRI (Fig 1F) CfoI (Fig 1G) DdeI(Fig 1H) PvuII RsaI (Fig 2E) and SspI H hordecaliswas distinguishedby Bsh1236I(Fig 1C) BsuRI (Fig 1F)MvaI (Fig 2C) and ScrFI (Fig 2F)
AluI AvaI CfoI HpaII MvaI RsaI and ScrFI sepa-rated the closely related and morphologically poorly dis-tinguished species from the H schachtii s str group(H schachtii H glycines H trifolii H medicaginis andH ciceri) from each other and all other species MvaI(Fig 2C) PvuII RsaI (Figs 2E 3) and ScrFI (Fig 2F)produced a speci c pattern for H schachtii which sep-arated this species from others Restrictions with MvaIRsaI HpaII or PvuII did not show intraspeci c variationin restriction patterns between the three H schachtii pop-ulations under study (Fig 3) Totalling the fragment sizesproduced by MvaI (Figs 2C 3) RsaI (Figs 2E 3) PvuII(Fig 3) or ScrFI (Fig 2F) yielded a sum of more than1060 bp indicatinga heterogeneityof ITS regions presentin the genome of H schachtii populations H glycinesanother member of the H schachtii s str group was dis-tinguished within this group by AvaI which produced apattern identical to that for H cajani (Fig 1B) H ca-jani was easily separated from others by many enzymesAluI (Fig 1A) Bsh1236I (Fig 1C) BsiZI (Fig 1D)BsuRI (Fig 1F) CfoI (Fig 1G) DdeI (Fig 1H) HinfIHpaII (Fig 2B) ScrFI (Fig 2F) TaqI (Fig 2H) andTru9I RFLP patterns produced by CfoI (Fig 1G) sep-arated H ciceri from others Heterogeneity of the ITSregion was observed for H ciceri with AluI (Fig 1A)CfoI (Fig 1G) RsaI (Fig 2E) Combinations of at leasttwo restriction enzymes separated other species from thisH schachtii s str group For example RsaI (Fig 2E) dis-tinguished H trifolii and H ciceri from other specieswhilst CfoI (Fig 1G) separated these two species RsaI(Fig 2E) demonstrated heterogeneity of ITS regions intwo populations of H trifolii and AluI in one populationof this species H medicaginis was distinguished fromother species by a combination of three enzymes HpaII(Fig 2B) MvaI (Fig 2C) and AvaI (Fig 1B Table 3)H salixophila belonging to the H schachtii group wasseparated from all species studied by Bsh1236I (Fig 1C)
156 Nematology
Identication of Heterodera species
Tab
le2
Num
ber
ofdi
ffer
entR
FL
Ppr
ole
s1 )yi
elde
dby
asi
ngle
enzy
me
dige
stio
nof
the
ITS
regi
ons
ofcy
stne
mat
odes
Spe
cies
Alu
IA
vaI
Bgl
IB
sh12
36I
Bsi
ZI
Bsp
143I
Bsu
RI
Cfo
ID
deI
Eco
RI
Hin
fIH
paII
Mva
IP
stI
Pvu
IIR
saI
Sal I
Scr F
ISf
u ISs
p ITa
q ITr
u 9I
Xba
I
Ha
vena
e(t
ype
A)
11
11
1a1
11
11
11
11
11
11
11
11
11
Ha
vena
e(t
ype
B)
21
11
11
11
11
11
11
12
11
11
11
1H
are
nari
a1
11
11
11
11
11
11
11
11
11
11
11
H
lipj
evi
21
11
11
21
11
21
12
12
11
11
22
1H
auc
klan
dica
21
11
11
12
11
11
11
12
11
11
31
1H
iri
21
12
22
13
11
22
21
12
12
11
32
1H
lat
ipon
s3
11
33
23
42
12
32
12
31
31
23
31
Hh
orde
cali
s4
11
43
14
11
12
33
11
11
41
14
11
Hs
chac
htii
52
15
41
55
32
34
41
1a4a
15a
11
31
1H
tri
foli
i5
5a2
15
41
55
32
32
51
34b
16
11
31
1H
med
icag
inis
52
15
41
55
32
34
51
34
15
11
31
1H
cic
eri
5a2
15
41
55a
32
32
51
34b
16
11
31
1H
sal
ixop
hila
23
16
51
66
41
45
11
15
71
32
15
41
Ho
ryzi
cola
61
17
63
77
51
55
63
46
17
31
65
1H
gly
cine
s5
41
54
15
53
23
45
13
41
51
13
11
Hc
ajan
i7
41
87
18
86
16
67
13
71
82
17
61
Hh
umul
i8
31
18
19
97
17
77
45
81
91
14
71
Hr
ipar
ia9
31
18
19
107
17
77
15
81
91
14
21
H
ci10
31
19
110
97
17
88
43
91
101
14
21
Hl
itor
alis
43
19
101
1111
81
77
94
310
111
11
42
1H
car
otae
111
210
114
1212
12a
91
72
75
111
112
11
48a
1H
cru
cife
rae
111
210
114
1212
91
72
75
111
112
11
48a
1H
eter
oder
asp
12
13
1112
513
1310
13
910
61
72
131
18
92
Hc
yper
i13
11
1213
614
1411
18
1011
76
123
144
19
101
Hg
oett
ingi
ana
141
213
114
1215
121
72
75
113
112
11
1011
1H
urt
icae
111
214
114
1212
91
73
75
111
11a
112
11
48
1M
eloi
dode
raal
ni15
11
1514
715
1613
19
1112
12a
11
141
151
34
31
1 )
num
bers
repr
esen
tid
enti
cal
rest
rict
ion
patt
erns
pa
tter
nsw
ith
aor
bha
vead
diti
onal
frag
men
ts
patt
erns
divi
ded
by
diff
ered
betw
een
popu
lati
ons
ofa
sing
lene
mat
ode
spec
ies
Vol 2(2) 2000 157
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
Digestion with 23 out of the 26 enzymes gave RFLPsfor all species studied BamHI HindIII and KpnI didnot restrict any of the ampli ed products No singleenzyme could distinguish all cyst forming species inthis study although some yielded much more taxonomicinformation than others (Table 2) For example CfoI(Fig 1G) yielded 16 polymorphic patterns distinguishing12 out of the 26 species AluI (Fig 1A) BsuRI (Fig 1F)Bsh1236I (Fig 1C) and ScrFI (Fig 2F) each produced15 polymorphic patterns and distinguished nine ten 11and nine species respectively The combination of thepatterns obtained by seven individual enzymes alloweddifferentiation of most species under study (Table 3) Insome cases the sum of restricted fragments length was lessthan 1060 bp This could be due to production of severalfragments with similar sizes orand of fragments smallerthat 100 bp which were poorly visible on the agarosegels Sometimes the sum of restricted fragment lengthswas more than the length of the unrestricted ampli edproduct Repeated digestion with an extended digestionperiod suggestedheterogeneityof ITS regionswas presentin the genome of these species
RFLP of ITS regions allowed clear differentiation ofmost agriculturally important cyst forming nematode spe-cies from each other and from their sibling species Somerestriction enzymes produced a RFLP pattern speci c fora species group For example the patterns obtained af-ter restriction with AluI (Fig 1A) Bsh1236I (Fig 1C)BsuRI (Fig 1F) DdeI (Fig 1H) HinfI (data not shown)and EcoRI (Fig 2A) distinguished the H schachtii s strgroup from the other groups whereas EcoRI restrictedonly ITS regions of species from this group BglI (datanot shown) BsiZI (Fig 1D) Bsp143I (Fig 1E) BsuRI(Fig 1F) and PstI (Fig 2D) separated the H goettin-giana group species having juveniles with four incisures(H carotae H cruciferae H goettingiana and H ur-ticae) from other species Some enzymes proved to be oflittle use for identi cation of larger numbers of the speciesbut were extremely adequate for individual species SspI(data not shown) for example only restricted PCR prod-ucts from H latipons and M alni whereas the ITS fromHeterodera sp was only cut by XbaI (data not shown)BsiZI (Fig 1D) and DdeI (Fig 1H) restricted the ITS re-gion of all species except H litoralis
As we observed earlier (Subbotin et al 1997) Eu-ropean populations of H avenae (type A) were easilyseparated from other species with AluI (Fig 1A) whichdigested the ITS regions of all studied nematodes ex-cept those of H arenaria No enzymes distinguished
European populations of H avenae from H arenariaH lipjevi was easily distinguished from all species byPstI (Fig 2D) BsuRI (Fig 1F) and TaqI (Fig 2H)which produced unique RFLP patterns for this speciesH aucklandica was separated by CfoI (Fig 1G) fromother species Bsh1236I (Fig 1C) CfoI (Fig 1G) andScrFI (Fig 2F) produceduniquepatterns for H iri H lati-pons was separated from the others by AluI (Fig 1A)Bsh1236I(Fig 1C) BsuRI (Fig 1F) CfoI (Fig 1G) DdeI(Fig 1H) PvuII RsaI (Fig 2E) and SspI H hordecaliswas distinguishedby Bsh1236I(Fig 1C) BsuRI (Fig 1F)MvaI (Fig 2C) and ScrFI (Fig 2F)
AluI AvaI CfoI HpaII MvaI RsaI and ScrFI sepa-rated the closely related and morphologically poorly dis-tinguished species from the H schachtii s str group(H schachtii H glycines H trifolii H medicaginis andH ciceri) from each other and all other species MvaI(Fig 2C) PvuII RsaI (Figs 2E 3) and ScrFI (Fig 2F)produced a speci c pattern for H schachtii which sep-arated this species from others Restrictions with MvaIRsaI HpaII or PvuII did not show intraspeci c variationin restriction patterns between the three H schachtii pop-ulations under study (Fig 3) Totalling the fragment sizesproduced by MvaI (Figs 2C 3) RsaI (Figs 2E 3) PvuII(Fig 3) or ScrFI (Fig 2F) yielded a sum of more than1060 bp indicatinga heterogeneityof ITS regions presentin the genome of H schachtii populations H glycinesanother member of the H schachtii s str group was dis-tinguished within this group by AvaI which produced apattern identical to that for H cajani (Fig 1B) H ca-jani was easily separated from others by many enzymesAluI (Fig 1A) Bsh1236I (Fig 1C) BsiZI (Fig 1D)BsuRI (Fig 1F) CfoI (Fig 1G) DdeI (Fig 1H) HinfIHpaII (Fig 2B) ScrFI (Fig 2F) TaqI (Fig 2H) andTru9I RFLP patterns produced by CfoI (Fig 1G) sep-arated H ciceri from others Heterogeneity of the ITSregion was observed for H ciceri with AluI (Fig 1A)CfoI (Fig 1G) RsaI (Fig 2E) Combinations of at leasttwo restriction enzymes separated other species from thisH schachtii s str group For example RsaI (Fig 2E) dis-tinguished H trifolii and H ciceri from other specieswhilst CfoI (Fig 1G) separated these two species RsaI(Fig 2E) demonstrated heterogeneity of ITS regions intwo populations of H trifolii and AluI in one populationof this species H medicaginis was distinguished fromother species by a combination of three enzymes HpaII(Fig 2B) MvaI (Fig 2C) and AvaI (Fig 1B Table 3)H salixophila belonging to the H schachtii group wasseparated from all species studied by Bsh1236I (Fig 1C)
156 Nematology
Identication of Heterodera species
Tab
le2
Num
ber
ofdi
ffer
entR
FL
Ppr
ole
s1 )yi
elde
dby
asi
ngle
enzy
me
dige
stio
nof
the
ITS
regi
ons
ofcy
stne
mat
odes
Spe
cies
Alu
IA
vaI
Bgl
IB
sh12
36I
Bsi
ZI
Bsp
143I
Bsu
RI
Cfo
ID
deI
Eco
RI
Hin
fIH
paII
Mva
IP
stI
Pvu
IIR
saI
Sal I
Scr F
ISf
u ISs
p ITa
q ITr
u 9I
Xba
I
Ha
vena
e(t
ype
A)
11
11
1a1
11
11
11
11
11
11
11
11
11
Ha
vena
e(t
ype
B)
21
11
11
11
11
11
11
12
11
11
11
1H
are
nari
a1
11
11
11
11
11
11
11
11
11
11
11
H
lipj
evi
21
11
11
21
11
21
12
12
11
11
22
1H
auc
klan
dica
21
11
11
12
11
11
11
12
11
11
31
1H
iri
21
12
22
13
11
22
21
12
12
11
32
1H
lat
ipon
s3
11
33
23
42
12
32
12
31
31
23
31
Hh
orde
cali
s4
11
43
14
11
12
33
11
11
41
14
11
Hs
chac
htii
52
15
41
55
32
34
41
1a4a
15a
11
31
1H
tri
foli
i5
5a2
15
41
55
32
32
51
34b
16
11
31
1H
med
icag
inis
52
15
41
55
32
34
51
34
15
11
31
1H
cic
eri
5a2
15
41
55a
32
32
51
34b
16
11
31
1H
sal
ixop
hila
23
16
51
66
41
45
11
15
71
32
15
41
Ho
ryzi
cola
61
17
63
77
51
55
63
46
17
31
65
1H
gly
cine
s5
41
54
15
53
23
45
13
41
51
13
11
Hc
ajan
i7
41
87
18
86
16
67
13
71
82
17
61
Hh
umul
i8
31
18
19
97
17
77
45
81
91
14
71
Hr
ipar
ia9
31
18
19
107
17
77
15
81
91
14
21
H
ci10
31
19
110
97
17
88
43
91
101
14
21
Hl
itor
alis
43
19
101
1111
81
77
94
310
111
11
42
1H
car
otae
111
210
114
1212
12a
91
72
75
111
112
11
48a
1H
cru
cife
rae
111
210
114
1212
91
72
75
111
112
11
48a
1H
eter
oder
asp
12
13
1112
513
1310
13
910
61
72
131
18
92
Hc
yper
i13
11
1213
614
1411
18
1011
76
123
144
19
101
Hg
oett
ingi
ana
141
213
114
1215
121
72
75
113
112
11
1011
1H
urt
icae
111
214
114
1212
91
73
75
111
11a
112
11
48
1M
eloi
dode
raal
ni15
11
1514
715
1613
19
1112
12a
11
141
151
34
31
1 )
num
bers
repr
esen
tid
enti
cal
rest
rict
ion
patt
erns
pa
tter
nsw
ith
aor
bha
vead
diti
onal
frag
men
ts
patt
erns
divi
ded
by
diff
ered
betw
een
popu
lati
ons
ofa
sing
lene
mat
ode
spec
ies
Vol 2(2) 2000 157
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
Identication of Heterodera species
Tab
le2
Num
ber
ofdi
ffer
entR
FL
Ppr
ole
s1 )yi
elde
dby
asi
ngle
enzy
me
dige
stio
nof
the
ITS
regi
ons
ofcy
stne
mat
odes
Spe
cies
Alu
IA
vaI
Bgl
IB
sh12
36I
Bsi
ZI
Bsp
143I
Bsu
RI
Cfo
ID
deI
Eco
RI
Hin
fIH
paII
Mva
IP
stI
Pvu
IIR
saI
Sal I
Scr F
ISf
u ISs
p ITa
q ITr
u 9I
Xba
I
Ha
vena
e(t
ype
A)
11
11
1a1
11
11
11
11
11
11
11
11
11
Ha
vena
e(t
ype
B)
21
11
11
11
11
11
11
12
11
11
11
1H
are
nari
a1
11
11
11
11
11
11
11
11
11
11
11
H
lipj
evi
21
11
11
21
11
21
12
12
11
11
22
1H
auc
klan
dica
21
11
11
12
11
11
11
12
11
11
31
1H
iri
21
12
22
13
11
22
21
12
12
11
32
1H
lat
ipon
s3
11
33
23
42
12
32
12
31
31
23
31
Hh
orde
cali
s4
11
43
14
11
12
33
11
11
41
14
11
Hs
chac
htii
52
15
41
55
32
34
41
1a4a
15a
11
31
1H
tri
foli
i5
5a2
15
41
55
32
32
51
34b
16
11
31
1H
med
icag
inis
52
15
41
55
32
34
51
34
15
11
31
1H
cic
eri
5a2
15
41
55a
32
32
51
34b
16
11
31
1H
sal
ixop
hila
23
16
51
66
41
45
11
15
71
32
15
41
Ho
ryzi
cola
61
17
63
77
51
55
63
46
17
31
65
1H
gly
cine
s5
41
54
15
53
23
45
13
41
51
13
11
Hc
ajan
i7
41
87
18
86
16
67
13
71
82
17
61
Hh
umul
i8
31
18
19
97
17
77
45
81
91
14
71
Hr
ipar
ia9
31
18
19
107
17
77
15
81
91
14
21
H
ci10
31
19
110
97
17
88
43
91
101
14
21
Hl
itor
alis
43
19
101
1111
81
77
94
310
111
11
42
1H
car
otae
111
210
114
1212
12a
91
72
75
111
112
11
48a
1H
cru
cife
rae
111
210
114
1212
91
72
75
111
112
11
48a
1H
eter
oder
asp
12
13
1112
513
1310
13
910
61
72
131
18
92
Hc
yper
i13
11
1213
614
1411
18
1011
76
123
144
19
101
Hg
oett
ingi
ana
141
213
114
1215
121
72
75
113
112
11
1011
1H
urt
icae
111
214
114
1212
91
73
75
111
11a
112
11
48
1M
eloi
dode
raal
ni15
11
1514
715
1613
19
1112
12a
11
141
151
34
31
1 )
num
bers
repr
esen
tid
enti
cal
rest
rict
ion
patt
erns
pa
tter
nsw
ith
aor
bha
vead
diti
onal
frag
men
ts
patt
erns
divi
ded
by
diff
ered
betw
een
popu
lati
ons
ofa
sing
lene
mat
ode
spec
ies
Vol 2(2) 2000 157
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
Fig 1 Restriction fragments of amplied ITS regions of cyst forming nematodes A AluI B AvaI C Bsh1236I D BsiZI E Bsp143IF BsuRI G CfoI H DdeI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
H humuli H riparia and H ci were differentiatedfrom other species and from each other by AluI (Fig 1A)Three other enzymes CfoI (Fig 1G) PstI (Fig 2D) andTru9I (Fig 4) separated H humuli from its sibling speciesH riparia H litoralis was distinguished by RFLP gen-erated by Bsh1236I (Fig 1C) BsiZI (Fig 1D) BsuRI
Inside the H goettingianagroup species were separatedby several enzymes Bsh1236I (Fig 1C) differentiated allspecies except H carotae and H cruciferae None ofthe enzymes separated these two species from each otherH goettingiana was distinguished from other species byAluI (Fig 1A) Bsh1236I (Fig 1C) CfoI (Fig 1G) DdeI
158 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
Identication of Heterodera species
Tab
le3
App
roxi
mat
esi
zes
ofre
stri
ctio
nfr
agm
ents
ofrD
NA
ITS
regi
ons
for
cyst
form
ing
nem
atod
es
Spe
cies
Alu
IA
vaI
Bsh
1236
IB
suR
IC
foI
Mva
IR
saI
Ha
vena
e(t
ype
A)
1060
1060
880
(500
380
)14
042
036
018
050
750
160
110
400
330
290
1040
Ha
vena
e(t
ype
B)
560
500
1060
880
140
420
360
180
5075
016
011
040
033
029
072
032
0H
are
nari
a10
6010
6088
014
042
036
018
050
750
160
110
400
330
290
1040
H
lipj
evi
560
500
1060
880
140
435
370
180
5075
016
011
040
033
029
072
032
0H
auc
klan
dica
560
500
1060
880
140
420
360
180
5075
020
011
040
033
029
072
032
0H
iri
560
500
1060
540
340
140
420
360
180
5041
034
016
011
042
033
029
072
032
0H
lat
ipon
s42
035
018
010
6088
016
053
051
075
011
042
033
029
090
016
0H
hor
deca
lis
880
180
1060
700
180
140
530
435
5075
016
011
044
033
029
010
40H
sch
acht
ii35
028
056
037
013
052
038
014
053
030
021
043
032
010
108
407
60
830
460
180
170
150
110
630
220
150
8038
0 23
0H
tri
foli
i(3
90)
350
280
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
060
023
018
017
0H
med
icag
inis
350
280
180
170
560
370
130
520
380
140
530
300
210
430
320
150
110
760
220
8083
023
0H
cic
eri
390
350
280
560
370
130
500
380
140
530
300
210
750
430
320
760
220
8083
060
023
018
017
015
011
0H
sal
ixop
hila
560
500
930
130
530
450
435
8020
016
015
040
033
029
077
029
0[1
060]
Ho
ryzi
cola
330
295
200
150
1010
320
270
200
130
360
210
8047
033
015
060
470
300
210
870
90H
gly
cine
s35
028
018
017
056
051
052
038
014
053
030
021
043
032
015
011
076
022
080
830
230
Hc
ajan
i36
020
018
056
051
047
038
014
053
031
012
032
027
016
015
076
030
010
6014
010
0H
hum
uli
460
250
180
170
930
130
880
140
450
110
5060
016
015
076
030
076
026
0H
rip
aria
630
250
180
930
130
880
140
450
110
5025
018
017
076
030
076
026
016
015
0H
ci
780
180
100
930
130
880
140
560
450
5060
016
015
069
020
080
560
480
Hl
itor
alis
880
180
930
130
670
390
610
450
350
310
250
150
560
310
290
240
800
260
Hc
arot
ae53
025
023
010
6083
014
070
530
330
170
480
270
(220
)76
030
060
033
013
017
011
0H
cru
cife
rae
530
250
230
1060
830
140
7053
033
017
048
027
017
011
076
030
060
033
013
0H
eter
oder
asp
45
040
024
010
6090
016
036
025
018
041
016
011
080
800
260
1060
150
90H
cyp
eri
410
360
200
160
1100
710
240
150
450
250
200
480
330
130
100
780
320
950
150
100
50H
goe
ttin
gian
a35
025
023
010
6083
023
053
033
017
028
027
019
011
076
030
048
021
013
012
0H
urt
icae
530
250
230
1060
830
120
530
330
170
480
270
170
110
760
300
600
(460
)33
013
0M
eloi
dode
raal
ni59
047
010
6041
014
053
030
023
040
035
016
010
0(7
80)
570
210
880
180
Ital
icle
tter
sad
diti
onal
frag
men
ts(
)ad
diti
onal
rest
rict
ion
frag
men
tsfo
rso
me
popu
lati
ons
[]
rest
rict
ion
for
som
epo
pula
tion
son
ly
Vol 2(2) 2000 159
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
Fig 2 Restriction fragments of amplied ITS regions of cyst forming nematodes A EcoRI B HpaII C MvaI D PstI E RsaI FScrFI G SfuI H TaqI (For species code see Table 1 lanes U unrestricted PCR product M 100 bp DNA ladder)
(Fig 1H) RsaI (Fig 2E) TaqI (Fig 2H) and Tru9I(Fig 4) H urticae parasite of nettle in many West Euro-pean countries differed from H carotae and H cruciferaeby RFLP patterns produced by three enzymes Bsh1236I(Fig 1C) HpaII (Fig 2B) and Tru9I (Fig 4) HpaIIclearly differentiated H urticae from these two species Itdid not digest the PCR product of H carotae and H cru-
ciferae but did restrict the ITS regions of H urticae pro-ducing two fragments (870 and 190 bp)
PCR ampli ed product obtained from H oryzicola andH cyperi differed from other species by its size Thedigestionwith different enzymes usually produced uniqueRFLP pro les for these two species (Figs 1 2 4 Table 3)The unidenti ed Heterodera sp from Cynodon dactylon
160 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
Identication of Heterodera species
Fig 3 Restriction fragments of amplied ITS regions of sugarbeet cyst nematode Heterodera schachtii (For species code seeTable 1 M 100 bp DNA ladder)
Fig 4 Restriction fragments of amplied ITS regions of cystforming nematodes digested by Tru9I (For species code seeTable 1 M 100 bp DNA ladder)
was also distinguished from all other species by severalenzymes (Table 2)
Intraspeci c variationwas revealed within some speciesAluI (Fig 1A) and RsaI (Fig 2E) distinguishedEuropeanpopulationsof H avenae(type A) from the Indian popula-tion (type B) These enzymes partly digested ITS regionsof three French populations indicating the heterogene-ity in rDNA (Subbotin et al 1997) Bsh1236I (Fig 1C)produced additional restriction fragments for the Rinkam(Bavaria) populationof H avenae These fragments how-ever were not found in other H avenae populations(Sub-botin et al 1999b)The two populationsof H salixophiladiffered by their RsaI restriction patterns This enzyme re-stricted the ITS regions of the Belgian populationbut not
that of the Ukrainian one (Table 3) Two populations ofM alni differed by the MvaI restriction pattern the en-zyme partly digested the ITS regions of the Belgian pop-ulation indicating a heterogeneity in this region of thespecies (Table 3) Intraspeci c variation in RFLP patternswas not revealed within H humuli H riparia (Subbotinet al 1997) H lipjevi (Subbotin et al 1999b) H iriH hordecalis H goettingianaand H schachtii
Discussion
The present work con rms that rDNA-RFLP allowclear differentiationof agriculturally important cyst form-ing nematode species from each other and from their sib-ling species RFLP produced by only seven enzymes sep-arated 21 species of cyst forming nematodes (Table 3)
Nematode species in the same genus are considered tohave similar sized ampli ed products of the spacer re-gions whereas size variation between genera is acceptedto be common (Powers 1996 1997) Our study demon-strated that the size of ampli ed ITS products is rather sta-ble within the genus Heterodera Only for H cyperi andH oryzicola did we obtain a deviating length so that thesespecies can easily be separated from other cyst formingnematode species Variation in the size of the ITS regionshas been reported for species from the genera Aphelen-choides (Ibrahim et al 1994) Meloidogyne (I De Leypers comm) and Pratylenchus (Orui 1996 L Waeyen-berge pers comm)
In the present study we used primers amplifying anrDNA product including the ITS1 ITS2 regions and the58S gene plus anking areas of the 18S and 28S genesPrimers used for ampli cation of nematode ribosomalDNA were recently reviewed (Fleming amp Powers 1998Powers amp Fleming 1998) For identi cation of some cystnematodes species Szalanski et al (1997) and Fleming etal (1998) used only the ITS1 region Although Ferris etal (1993 1994) and Blok et al (1998) all studying cystforming nematodes reported more variation in the ITS1region than in the ITS2 the use of both these regions looksto be more promisingbecause after its digestion the largersize of the PCR ampli ed fragment yields more informa-tive patterns Moreover some restriction sites useful foridenti cation of several agricultural important species aresituated in the ITS2 region (unpubl)
ITS regions are considered to be rather conservativeand so not appropriate for separation of populations be-longing to same species However intraspeci c variationsin these regions have been revealed within plant parasitic
Vol 2(2) 2000 161
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
nematode populations of H zeae and H trifolii (Szalan-ski et al 1997) H avenae (Bekal et al 1997 Subbotinet al 1999b) and G pallida (Blok et al 1998) Dif-ferences in RFLP between populations can be presentedas the existence of differences in restriction sites in ITSsequence andor the appearance of additional ITS hap-lotypes with different sequences Heterogeneity in ITSregions or presence of several ITS haplotypes within asingle genome were found during the present work forpopulations of H avenae H ciceri H carotae H cru-ciferae H schachtii H trifolii H urticae Heterodera spand M alni ITS heterogeneity was reported for Meloido-gyne (Zijlstra et al 1995) Belonolaimus (Cherry et al1997) Radopholus (L Waeyenberge pers comm) andfor several cyst forming nematode species H zeae (Sza-lanski et al 1997) G pallida (Blok et al 1998) andG rostochiensis (Subbotin et al unpubl) and perhapsis widely distributed among nematodes The mechanismsupporting such a mixture of haplotypes in one genome isnot clear
Relationshipsbetween such changes in rDNA and chan-ges in the morphological and biological peculiarities ofpopulations has not been studied in detail yet The Indianpopulation of H avenae (ITS type B) used in our studydiffered from most European populations by RFLP ob-tained by two restriction enzymes and can also be sepa-rated by morphometrics Several French populations be-longing to different groups of pathotypes contain a mix-ture of two ITS types (A+ B) (Subbotin et al 1999b)Molecular polymorphism has frequently been observedbetween geographicallyisolated populationsSzalanski etal (1997) reported differences in H trifolii from the USAand Australia and in H zeae from the USA and Indiaas opportunities for gene exchangebetween these popula-tions were restricted Differences in ITS sequences wereobserved in two populationsof H latipons one from Ros-tov region Russia and one from Gilat Israel (V Ferris etal unpubl) When using several restriction enzymes thesenior author was able to separate the Rostov populationfrom a Syrian H latipons population For example RsaIand PvuII did not digest the ITS regionsof the Syrian pop-ulation (Subbotin et al unpubl) Bekal et al (1997) alsoreported that RsaI did not restrict ITS regions of popu-lations from Israel and Syria It is obvious that the taxo-nomic status the Rostov population of H latipons needsclari cation
The Heterodera sp sampled for the present study fromthe Ukraine belongs to the H cardiolata complex Thiscomplex contains the morphologicallyclosely related spe-
cies H cardiolata H graminis and H cynodontis all in-fecting Cynodondactylon and found in various regions ofthe world Australia South Africa Fiji Islands TrinidadIndia Tadzhikistan Uzbekistan and Pakistan (Kirjanovaamp Ivanova 1969 Stynes 1971 Luc 1986 Shahina ampMaqbool 1993) Perhaps some of these species can beconsidered as synonyms or as geographical subspeciesof H cardiolata However we found that two (AluI andBsuRI) from the nine studied enzymes produced differentRFLP pro les for the Heterodera sp from the Ukraineand H cynodontis from Pakistan (unpubl)More detailedmorphological and molecular studies of this species com-plex are needed for understanding the relationships be-tween the populationsand supporting the validity of someof these species
We did not nd restriction enzymes that enable the sep-aration of H avenae from H arenaria and H carotaefrom H cruciferae proving that these species are closelyrelated These species can only be distinguishedfrom eachother by minor morphometrical and morphological char-acteristics (Baldwin amp Mundo-Ocampo 1991 Robinsonet al 1996)
The ampli cation and analysis of the ITS has a lot ofadvantages The rapidity to obtain pro les and the clar-ity of the results allow identi cation of most species veryeasily This technique is relatively easy to operate andnot expensive It is particularly suited for determining theidentity of quarantine nematodes where it is often neces-sary to perform identi cation on very few individualspec-imens but where an incorrect identi cation can have ma-jor economical implications (Vrain amp McNamara 1994Szalanski et al 1997) However in order to be able to ap-ply the techniques as a routine in quarantine inspectionsor plant protection services it is necessary to make a cat-alogue of RFLP in the ITS region of widely distributednematode species
Acknowledgements
The senior authorgratefully acknowledgesthe nancialsupport of a NATO Research Fellowship The authorsthank Drs M Bossis VN Chizhov J Grunder L Naso-nova of a RT Robbins M Romero J Rowe B Schoe-maker D Sturhan N Vovlas and W Wouts for supplyingnematode populations
162 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
Identication of Heterodera species
References
BALDWIN JG amp MUNDO-OCAMPO M (1991) Heteroderi-nae cyst- and non- cyst-forming nematodes In Nickle WR(Ed) A manual of agricultural nematology New York NYUSA Marcel Dekker Inc pp 275-362
BEKAL S GAUTHIER JP amp RIVOAL R (1997) Geneticdiversity among a complex of cereal cyst nematodes inferredfrom RFLP analysis of the ribosomal internal transcribedspacer region Genome 40 479-486
BLOK VC MALLOCH G HARROWER B PHILLIPS MS amp VRAIN TC (1998) Intraspecic variation in ribo-somal DNA in populations of the populations of the potatocyst nematode Globodera pallida Journal of Nematology 30262-274
CHERRY T SZALANSKI AL TODD TC amp POWERSTO (1997) The internal transcribedspace region of Belono-laimus (Nemata Belonolaimidae)Journal of Nematology 2923-29
EVANS K amp ROWE JA (1998) Distribution and economicimportance In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 1-30
FERRIS VR FERRIS JM amp FAGHIHI J (1993) Variationin spacer ribosomal DNA in some cyst-forming species ofplant parasitic nematodes Fundamental and Applied Nema-tology 16 177-184
FERRIS VR FERRIS JM FAGHIHI J amp IREHOLM A(1994) Comparisons of isolates of Heterodera avenae using2-D PAGE protein patterns and ribosomal DNA Journal ofNematology 26 144-151
FLEMING CC amp POWERS TO (1998) Potato cyst nema-tode diagnostics morphology different hosts and biochem-ical technique In Marks RJ amp Brodie BB (Eds) Potatocyst nematode Biology distributionand control WallingfordUK CAB International pp 91-114
FLEMING CC TURNER SJ POWERS TO amp SZALAN-SKY AL (1998) Diagnostics of cyst nematodes use of thepolymerase chain reaction to determine species and estimatepopulation level Aspect of Applied Biology 52 375-382
IBRAHIM SK PERRY RN BURROWS PR amp HOOPER DJ (1994) Differentiation of species and populations ofAphelenchoides and of Ditylenchus angustus using a frag-ment of ribosomal DNA Journal of Nematology 26 412-421
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 StPatrickrsquos College Maynooth Co Kildare Ireland Luxem-bourg European Commission DG XII pp 178-187
KIRJANOVA ES amp IVANOVA TS (1969) [A cyst-forming nematode Heterodera cardiolata n sp (Nema-
toda Heteroderidae) from Dushanbe Tadzhikistan]DokladyAkademii Nauk Tadzhikskoi SSR 12 59-62
LUC M (1986) Cyst nematodes in equatorial and hot tropicalregions In Lamberti F amp Taylor CE (Eds) Cyst nema-todes New York amp London Plenum Press pp 355-372
MATHEWS HJP (1971) Morphology of the nettle cyst nema-tode Heterodera urticae Cooper 1955 Nematologica (1970)16 503-510
ORUI Y (1996) Discrimination of the main Pratylenchusspecies (Nematode Pratylenchidae) in Japan by PCR-RFLPanalysis Applied Entomology and Zoology 31 505-514
ORUI Y (1997) [Discrimination of Globodera rostochiensisand four Heterodera species (Nematoda Heteroderidae) byPCR-RFLP analysis] Japanese Journal of Nematology 2767-75
POWERS TO (1996) Molecular diagnosticsof plant and insectparasitic nematodes In Marshall G (Ed) Proceedings ofdiagnostics in crop production symposium Farnham UKBritish Crop Production Council pp 121-126
POWERS TO (1997) The rDNA internal transcribed spacerregion as a taxonomic marker for nematodes Journal ofNematology 29 441-450
POWERS TO amp FLEMING CC (1998) Biochemical andmolecular characterization In Perry RN amp Wright DJ(Eds) The physiology and biochemistry of free-living andplant-parasitic nematodes Wallingford UK CABI Publish-ing pp 355-380
ROBINSON AN STONE AR HOOPER D amp ROWE JA(1996) A redescriptionof Heteroderaarenaria Cooper 1955a cyst nematode from marram grass Fundamental and Ap-plied Nematology 19 109-117
SHAHINA F amp MAQBOOL MA (1995) Cyst nematodes ofPakistan (Heteroderidae) Karachi Pakistan University ofKarachi 155 pp
STYNES BA (1971) Heterodera graminis n sp a cyst nema-tode from grass in Australia Nematologica 17 213-218
SUBBOTIN SA STURHAN D WAEYENBERGE L ampMOENS M (1997) Heterodera riparia sp n (TylenchidaHeteroderidae) from common nettle Urtica dioica L andrDNA-RFLP separation of species from the H humuli groupRussian Journal of Nematology 5 143-157
SUBBOTIN SA HALFORD PD amp PERRY RN (1999a)Identi cation of populations of potato cyst nematodesfrom Russia using protein electrophoresis rDNA-RFLP andRAPDs Russian Journal of Nematology 7 57-63
SUBBOTIN SA WAEYENBERGE L MOLOKANOVA IAamp MOENS M (1999b) Identi cation of Heterodera avenaegroup species by morphometrics and rDNA-RFLPs Nema-tology 1 195-207
SZALANSKI A SUI DD HARRIS TS amp POWERS TO(1997) Identi cation of cyst nematodes of agronomic andregulatory concern with PCR-RFLP of ITS1 Journal ofNematology 29 255-267
Vol 2(2) 2000 163
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
164 Nematology
SA Subbotin et al
THIEacuteRY M amp MUGNIEacuteRY D (1996) Interspecic rDNArestriction fragment length polymorphism in Globoderaspecies parasites of solanaceous plants Fundamental andApplied Nematology 19 471-479
VRAIN TC amp MCNAMARA DG (1994) Potential for iden-ti cation of quarantine nematodes by PCR EPPO Bulletin24 453-458
VRAIN TC WAKARCHUK DA LEVESQUE AC ampHAMILTON RI (1992) Intraspecic rDNA restriction frag-ment length polymorphisms in the Xiphinema americanumgroup Fundamental and Applied Nematology 15 563-574
WENDT KR VRAIN TC amp WEBSTER JM (1993) Sep-aration of three species of Ditylenchus and some host ra-
ces of D dipsaci by restriction fragment length polymor-phism Journal of Nematology 25 555-563
WOUTS WM amp BALDWIN JG (1998) Taxonomy andidenti cation In Sharma SB (Ed) The cyst nematodesLondon UK Kluwer Academic Publishers pp 83-122
WOUTS WM amp STURHAN D (1995) Heterodera auck-landica sp n (Nematoda Heteroderidae) from a NewZealand native grass with notes on the species of the H ave-nae group New Zealand Journal of Zoology 22 199-207
ZIJLSTRA C LEVER AEM UENK BJ amp VAN SILF-HOUT CHY (1995) Differencesbetween ITS regionsof iso-lates of root-knot nematodes Meloidogyne hapla and M chit-woodi Phytopathology 85 1231-1237
