Nema ology, 1999, Vol. 1(3), 321-332
Isola ion and cha ac e isa ion o He e o habdi is spp. (Nema oda:
He e o habdi idae) om Hunga y, Es onia and Denma k
Ch is ine T. GRIFFIN, Ilona DIX, Susan A. JOYCE, Ann M. BURNELL and Ma in J. DOWNES
Depa men o Biology, Na ional Uni e si y o I eland, Maynoo h, Co. Kilda e, I eland
Accep ed o publica ion:31 May 1998
Summa y – Ta ge ed su eys we e conduc ed o he en omopa hogenicnema ode He e o habdi is in a eas o Denma k, Es onia and
Hunga y.Isola eswe e iden i ed by IEF, PCR and c oss- e ili y es sas belonging o h ee dis inc axonomicg oups: H. bac e iopho a,
he no h-wes Eu opean (NWE) ype o H. megidis and he I ish ype o He e o habdi is. The I ish and NWE ypes o He e o habdi is
we e bo h p esen in Denma k (a six and ou si es, espec i ely),while only he NWE ype was eco e edin Es onia. H. bac e iopho a
was he dominan he e o habdi ididen i ed in Hunga y ( en si es),bu he I ish ype was also de ec eda wo si es. This is he  s epo
o he I ish ype o He e o habdi is on con inen al Eu ope. Co-occu ence o wo He e o habdi is ypes a a single si e was no ed in
Denma k (I ish and NWE) and in Hunga y (I ish and H. bac e iopho a). He e o habdi is was eco e ed a 38.5% o si es (n = 26) in
Denma k (no h coas o Sjælland), 27.3% o he coas al si es (n = 22) in Es onia, and 32.6% o si es (n = 46) in Hunga y.
Résumé – Isola ion e ca ac é isa ion d’espèces d’He e o habdi is (Nema oda: He e o habdi idae) o iginai es de Hong ie,
d’Es onie e du Danema k –Des p ospec ionsciblées on é é e ec uées dans ce aines égions du Danema k, d’Es onie e de Hong ie
pou eche che les néma odes du gen e He e o habdi is. Les souches, iden i ées pa les mé hodes de concen a ion isoélec ique, de
PCR e d’hyb ida ion, appa iennen aux ois g oupes axinomiques d’He e o habdi is:H. bac e iopho a, le g oupe de l’Eu ope du
no d-oues (NWE) de H. megidis e le g oupe i landais d’He e o habdi is. Le g oupe i landais e le g oupe NWE son ous les deux
p ésen s au Danema k (dans six e qua e si es, espec i emen ),maisseul lede nie g oupea é é encon éen Es onie. H. bac e iopho a,
p ésen dans dix si es, es l’espèce dominan e d’He e o habdi is en Hong ie, mais le g oupe i landaisa é é égalemen dé ec é dans deux
si es. C’es la p emiè e ois que le g oupe i landais es encon é en Eu ope con inen ale. La p ésence simul anée de deux ypes d’He-
e o habdi is es signalée au Danema k (g oupe i landais e g oupe NWE) e en Hong ie (g oupe i landais e H. bac e iopho a). Des
He e o habdi is on é é collec és su 38.5% des 26 si es du Danema k (cô e no d de Sjælland), 27.3% des 22 si es du li o al es onien
e 32.6% des 46 si es de Hong ie.
Keywo ds: biologicalcon ol, en omopa hogenicnema ode, geog aphicdis ibu ion,iden ica ion, soil su ey.
En omopa hogenic nema odes o he amilies He e o-
habdi idae and S eine nema idae ha e conside able po-
en ial o he con ol o insec pes s. The non- eeding
in ec i e ju enile (IJ) can su i e in he soil o se e al
mon hs un il suscep ible insec s a e encoun e ed. The IJs
en e insec s and, oge he wi h hei symbio ic bac e ia,
kill hem wi hin days.
Su eys o en omopa hogenic nema odes ha e been
conduc edin many pa so he wo ld, includingAus alia
(Akhu s & Bedding, 1986), he Uni ed S a es (Akhu s
& B ooks, 1984; Ha a e al., 1991) and Eu ope (M ácek,
1980; Deseö & Mille , 1985; Bu man e al., 1986; Black-
shaw, 1988; Vänninen e al., 1989; Hominick & B iscoe,
1990; G i  n e al., 1991; 1994b), o he pu poses bo h
o eco e ing po en ially use ul isola es and o gaining an
insigh in o he ecology o he nema odes.
Smi s e al. (1991) iden ied He e o habdi is isola es
om Eu ope, on he basis o es ic ion leng h polymo -
phisms, as being o h ee ypes: He e o habdi is bac e-
iopho a, he no h-wes Eu opean (NWE) g oup and he
I ish g oup. Cu en e idence sugges s ha he H. bac e-
iopho a g oup is he dominan He e o habdi is in sou h-
e n and cen al Eu ope. Thus, Smi s e al. (1991) iden i-
 ed isola es om I aly, Spain, cen al Ge many (Da m-
s ad ), and Moldo a as H. bac e iopho a, and he species
has also been isola ed in he sou h o F ance (G enie
e al., 1996). M ácek and Jense (1988) iden i ed a he -
e o habdi id isola ed nea Budapes as H. helio hidis,
which is now conside ed o be conspecic wi h H. bac-
e iopho a (Poina , 1990). Isola es om he Ne he lands,
Poland and he no h o Ge many we e assigned by Smi s
e al. (1991) o he NWE ype o He e o habdi is. This
ype has also been isola ed in he sou h o England (Ho-
minick e al., 1995) and in Belgium (Midu u i e al.,
1996). While mos o he eco ds o i ha e been om
no he n Eu ope, he ype was also eco e ed in no h-
c
®Koninklijke B ill NV, Leiden, 1999 321
C.T. G i  n e al.
e n G eece (Men i e al., 1997). The I ish ype o He e-
o habdi is is, o da e, he only he e o habdi id o ha e
been isola ed in I eland, whe e i is ela i ely common in
sandy coas al g asslands(G i  n e al., 1994a).I was also
ound associa ed wi h coas al si es in B i ain (G i  n e
al., 1994b; Hominick e al., 1995), bu has no p e iously
been eco ded ou side o hese islands.
The NWE ype o He e o habdi isand H. megidis sha e
he same DNA es ic ion p o les o all he es ic ion
enzymes es ed by Joyce e al. (1994a). The e is s ong
e idence, howe e , ha he I ish ype ep esen s a sepa-
a e species. I ish ype isola es possess a dis inc i e IEF
p o ein elec opho eg am(Joyce e al., 1994b) and a dis-
inc i e epe i i e DNA es ic ion p o le (Smi s e al.,
1991). Also, hey can be dis inguished om NWE ype
He e o habdi is by m DNA and DNA ITS es ic ionp o-
 les o selec ed enzymes (Joyce e al., 1994a), and hey
a e ep oduc i ely isola ed om H. megidis and NWE
isola es (Dix e al., 1992; G i  n e al., 1994b; Joyce e
al., 1994b). Adams e al. (1998) sequenced 716 bases o
he DNA ITS1 egion o nine pu a i e species o He e-
o habdi is and obse ed 35 nucleo ide subs i u ions be-
ween he I ish ype isola e K122 and H. megidis, wo o
which a e au apomo phies o K122. The e is hus cumu-
la i e e idence ha H. megidis and NWE ypea e conspe-
ci c bu he I ish ype is a dis inc species.
He e we epo he esul s o a sampling p og amme
in Hunga y, Denma k and Es onia. The e a e no p e ious
epo s o en omopa hogenic nema odes om Denma k
o Es onia. In Hunga y, limi ed sampling in he icin-
i y o Budapes eco e ed a single H. bac e iopho a iso-
la e (M ácek & Jense , 1988). The p esen sampling p o-
g amme was pa o a b oade sea ch in Eu ope aimed a
he eco e y o no el wild ype s ains o He e o habdi is
wi h use ul ai s. The e o e, sampling was di ec ed a
si es a which i was conside ed likely ha he genus was
p esen , iz sandy soils, and especiallycoas al si es, which
we (G i  ne al.,1994b)ando he s(e.g. Ha a e al., 1991;
Ama asinghe e al., 1994) ha e ound o be a sui able
habi a o he e o habdi ids.
Ma e ials and me hods
SURVEY AND SAMPLING
Po en ial sampling si es in Denma k, Es onia and Hun-
ga y we e ini ially iden ied om maps and geologi-
cal da a and subsequen ly by local inspec ion. Si es in
Denma k and Es onia we e all coas al (Fig. 1A, B). In
Denma k, sampling was es ic ed o he no h coas o
Sjælland (Fig. 1A). Sampling in Hunga y was conduc ed
in wo egions whe e soils a e ela i ely sandy, nea
Kecskemé and Deb ecen, espec i ely (Fig. 1C, D). Be-
ween he i e s Danube and Tisza, in and a ound he
Kiskungsági Na ional Pa k (sou h o Kecskemé ; Bács-
Kiskun coun y), he e a e ex ensi e sand hills consis ing
o i e deposi ed sand ewo ked by he wind. The second
a ea in Hunga y was o he no h and eas o Deb ecen
(Hajdú-Biha and Szabolcs-Sza má coun ies).
Soil sampling and bai ing wi h Galle ia mellonella
we e as desc ibed by G i  n e al. (1994b) wi h he
addi ion ha samples ound o be posi i e o He e o-
habdi is we e bai ed a second ime. The main sampling
p og amme was conduc ed in Oc obe -No embe , 1991.
Ta ge ed sampling o con m some o he o iginal  nd-
ings was ca ied ou in Sjælland in Decembe , 1992 and
in Hunga y in June, 1994.
MOLECULAR CHARACTERISATION
The isola es om he main sampling p og amme we e
iden i ed om p o ein isoelec ic ocusing elec opho e-
g ams (pH ange 3-10) o soluble p o eins om IJ as de-
sc ibed by Joyce e al. (1994b). This diagnosis was sub-
sequen ly con med by es ic ion enzyme diges o he
in e nal ansc ibed space (ITS) egion o DNA as de-
sc ibed by Joyce e al. (1994a), using he MboI es ic-
ion enzyme. Isola es om la e a ge ed sampling p o-
g ammes we e cha ac e ised using DNA ITS es ic ion
p o les only.
In he case o isola es exp essing he HP88 es ic ion
p o le, he DNA non ansc ibed space (NTS) egion
was ampli ed as desc ibed by Joyce e al. (1994c) and he
ca 1.4 kb ampli ca ion p oduc DNA was diges ed using
he enzymes HaeIII and HpaII.
CROSS-BREEDING STUDIES
In e s ain c osses we e pe o med on lipid aga pla es
as desc ibed by Dix e al. (1992). A leas 60 second
gene a ion i gin emales we e used o each in e s ain
c oss. Re e ence s ains used o he iden ica ion o
newly isola ed nema odesa e shown in Table 1.
Resul s
He e o habdi is was isola ed in Sjælland (Denma k),
Es onia and Hunga y. The e was no di e ence be ween
322 Nema ology
He e o habdi isspp. om Hunga y, Es onia and Denma k
Fig. 1. Si es sampled o He e o habdi is. A: Es onia; B: Sjælland, Denma k; C: Kecskemé egion (Bács-Kiskun coun y), Hunga y and
D: Deb ecen egion (Hajdú-Biha and Szabolcs-Sza má coun ies), Hunga y. (Each symbol may ep esen mo e han one si e; scale
ba =10 km).
Vol. 1(3), 1999 323
C.T. G i  n e al.
Table 1. O igin and sou ce o e e ence s ains o He e o habdi is.
Name O igin ITS es ic ion p o le Sou ce
HB1 B econ, Aus alia H. bac e iopho a R.J. Akhu s *
HP88 U ah, USA H. bac e iopho a HP88 P. Wes e man**
Da ms ad Da ms ad , Ge many H. bac e iopho a HP88 T. Jackson***
K122 Wex o d, I eland I ish ype Own collec ions
HF85 Fle opolde ,The Ne he lands H. megidis NWE ype P. Wes e man**
*CSIRO, Canbe a, Aus alia.
** Van Hall Ins i u e, Leeuwa den, The Ne he lands.
*** New Zealand Pas o al Ag icul u e Resea ch Ins i u e L d., Lincoln, New Zealand.
Fig. 2. MboI es ic ion diges s o he PCR ampli ca ion p oduc s o he DNA in e nal ansc ibed space egion o He e o habdi is
isola es, sepa a ed on a 2% aga ose gel. 1: EU333; 2: EU335; 3: EU347; 4: EU348; 5: EU349; 6: EU94; 7: EU106; 8: K122; 9:
EU85; 10: HF85; 11: EU339; 12: EU362; 13: EU367; 14: EU368; 15: EU369; 16: HP88; 17: Da ms ad ; 18: HB1; M: 1 kb ma ke .
S ains EU85 (lane 9) and EU106 (lane 7) a e om Denma k, all o he s ains wi h he EU designa ion a e om Hunga y.
he h ee coun ies in he p opo ion ei he o si es o
o samples posi i e o He e o habdi is (Table 2). O e -
all, he genus was eco e ed a 33% o si es, and in
16% o samples. Th ee species we e de ec ed (Table 2):
H. megidis NWE ype in Denma k and Es onia; I ish ype
He e o habdi is in Denma k and Hunga y, and H. bac e-
iopho a (HP88 ype es ic ion p o le) in Hunga y. The
MboI es ic ion p o les o he DNA ITS egion om a
ep esen a i esample o hese isola esa e shown in Fig. 2.
The ain bands isible on his gel ep esen incomple e
diges ionp oduc s.
SJÆLLAND (DENMARK)AND ESTONIA
All o he si es sampled in Sjælland and Es onia (and
hence all o he si es posi i e o He e o habdi is) we e
wi hin 1.5 km o he sea; he majo i y we e wi hin 200 m
o i . In Es onia, He e o habdi is was de ec ed a six o
he 22 si es sampled, and all isola es we e iden ied as
he NWE ype (Table 2; Fig. 1A). Posi i e si es included
meadow, ec ea ional g assland a eas, open coni e ous
o es wi h ex ensi e g ass co e and he ma gins o a
ce eal  eld (Table 3).
Bo h he I ish ype and NWE ype o He e o habdi is
we e isola ed in Sjælland (Table 2; Fig. 1B). Mos o
he si es om which He e o habdi is was eco e ed we e
dune o u ec ea ional a eas, o en wi h sh ubs and/o
coni e s p esen (Table 3). Bo h I ish and NWE ypes oc-
cu ed oge he a one si e, Gudmind up Lyng. Six sam-
ples we e aken a his si e; he NWE and I ish ypes
we e each eco e ed om one sample. The I ish ype
324 Nema ology
He e o habdi isspp. om Hunga y, Es onia and Denma k
Table 2. Occu ence o h ee ypes o He e o habdi is (H. bac e iopho a [H.b] G oup, he No h Wes Eu opean ype o H. megidis
[H.m] and I ish ype He e o habdi is) a si es in Hunga y, Denma k and Es onia, Oc obe -No embe 1991.
Coun y Numbe o Numbe (& %) Numbe o si es wi h He e o habdi is
si es and o si es and iden ied o species
samples samples wi h To al H.b H.m I ish
He e o habdi is
Es onia 22 6 (27.3%) a6 0 6 0
81 9 (11.1%) a
Sjælland 26 10 (38.5%) a9 0 4 6
(Denma k) 85 15 (17.6%) a
Hunga y
Kecskemé 22 10 (45.5%) A8 6 0 2
39 12 (30.8%) A
Deb ecen 24 5 (20.8%) A4 4 0 0
57 6 (10.5%) B
To al 46 15 (32.6%) a12 10 0 2
96 18 (18.8%) a
En ies ollowed by he same le e do no di e signi can ly (P<0.05) om each o he . Lowe case le e s: be ween coun ies
(chi-squa e es ); uppe case le e s: be ween egions o Hunga y (Fishe ’s exac es ). No mal le e s: si es; bold le e s: samples.
was eco e ed om he landwa d side o a dune idge,
wi h mainly sh ubs (Rosa ugosa,Empe um nig um and
hea he s) and some g ass, while he NWE ype came
om a g ass/clo e oadside e ge bo de ing ha a ea.
An examina ion o he si e desc ip ions o o he He-
e o habdi is-posi i e si es in Sjælland sugges ed a pos-
sible associa ion be ween he p esence o I ish He e o-
habdi is and Rosa ugosa. ExcludingGudmind upLyng,
whe e bo h nema ode ypes occu ed, oses we e p esen
a ou o he  e I ish-posi i e si es, bu a none o
he h ee NWE-posi i e si es. A es o associa ion be-
ween nema ode ype and p esence o oses a nema ode-
posi i e si es was signi can a P= 0.07 (Fishe ’s exac
es ).
HUNGARY
H. bac e iopho a was he dominan ype p esen in
Hunga y, bu he I ish ype was iden i ed a wo si es in
he Kecskemé egion (Table 3; Fig. 1C). He e o habdi is-
posi i e si es in Kecskemé included meadow, pas u e, a
g assy hillside wi h junipe , a ye  eld wi h associa ed
e ge, a wooded oadside e ge, and he bank o a d ied-
up alkaline lake (Table 3). In he Deb ecen a ea, only
H. bac e iopho a was de ec ed (Fig. 1D). Si es in his
egion had he hea ies soils o all hose sampled in any
o he h ee coun ies, being sandy loams o sandy clay
loams a he han sands. Posi i e si es he e we e pas u e,
was e g assland and he ma ginso a maize  eld (Table 2).
O e all, one hi d o he si es sampled in Hunga y we e
posi i e o He e o habdi is (Table 2). He e o habdi is
was de ec ed a a highe p opo iono si es in he Kecske-
mé egion (46%) han in he Deb ecen egion (21%), and
he di e ence was signi can a P= 0.054 (Fishe ’s
exac es ). The di e ence be ween hese wo egions
o Hunga y eached signicance a P<0.05 when
he p opo ion o posi i e samples was compa ed (31 s
11%) (Table 2). When only he da a o H. bac e iopho a
(which occu ed in bo h egions) we e conside ed, he
p e alence emained highe in Kecskemé (6/22 si es,
6/39 samples) han Deb ecen (4/24 si es, 4/59 samples)
bu he di e ence was no signi can o ei he si es o
samples.
In a es ic ed sampling p og amme in June 1994, se -
e al soil samples we e aken a each o he wo Kecskemé
si es a which he I ish ype He e o habdi is isola es
(EU167 and EU176) had been eco e ed in 1991. These
we e espec i ely, a wide oadside e ge wi h hedge ow
b oadening o deciduouswoodland, and a low-lying pas-
u e wi h eeds (Ph agmi es aus alis) adjoining he sam-
pled a ea. I ish ype He e o habdi is was again isola ed in
Vol. 1(3), 1999 325

C.T. G i  n e al.
Table 3. Loca ions o si es whe e He e o habdi is was de ec ed in Oc obe -No embe 1991, he habi a om which posi i e samples
we e aken, and he iden i y o he isola ed He e o habdi is (H.m.: H. megidis NWE ype; H.b.: H. bac e iopho a; I ish: I ish ype o
He e o habdi is; He .: iden ied o genus only).
Si e loca ion Sample/isola e Iden i y Habi a a sample
code o isola e
Es onia
Tallinn EU6 H.m. Pa k: u wi h Elymus
Keile Joa EU17 H.m. Open coni . o es wi h g ass co e
Lohusalu EU21 H.m. Meadow
Taagupi EU36 H.m. G ass
EU38 H.m. Weedy ma gin o ce eal  eld
Aa EU70 H.m. Tu g ass
EU71 H.m. G assy slope
Tsi e EU80 Meadow
EU81 He . Meadow
Denma k: Sjælland
Mø d up EU85 H.m. G ass (was e g ound)
Ho nbæk EU94 I ish Open coni e ous o es wi h g ound oak,
oses and g ass
Gilleleje EU103 He . G assy slope
EU106 ** I ish Sh ubs and long g ass
Rågeleje EU114 I ish Meadow
W. o Rågeleje EU116 I ish G ass
EU121 I ish G assy slope
Liseleje EU132 I ish Dunes, some oses
EU133 He . Dunes, some oses
Ny up EU140 H.m. Long g ass, sca e ed coni e s
EU141 He . Long g ass, sca e ed coni e s
Lumsas EU143 He . G ass (pa king place)
Gudmind up Lyng EU158 I ish G ass/ ose/hea he /Vaccinium
EU162 H.m. G ass e ge
Vindekilde EU166 H.m. Old dunes, long g ass
Hunga y: Kecskemé
Nea Kecskemé EU167 ** Roadside e ge wi h ees
*** I ish
*KNP IV EU173 *** He . Sho g ass, bank o d y alkaline lake
nea KNP V EU175 He . Pas u e
KNP V(1) EU176 *** I ish Pas u e
KNP V(2) EU178 H.b. Rye  eld including e ge
KNP V(3) EU185 H.b. Sand hills; g ass and junipe
KNP V(4) EU188 H.b. Meadow on hillside
EU190 He . Pas u e
KNP V(5) EU193 H.b. Meadow
KNP V(6) EU195 H.b. Meadow
EU196 He . Meadow
KNP VI (Bugac) EU201 H.b. Cu pas u e
326 Nema ology
He e o habdi isspp. om Hunga y, Es onia and Denma k
Table 3. (Con inued).
Si e loca ion Sample/isola e Iden i y Habi a a sample
code o isola e
Hunga y: Deb ecen
Lé a é es EU222 H.b. Sheep pas u e
Nyí bél ek EU228 H.b. Pas u e on hill
EU229 He . Pas u e
Téglás EU246 H.b. G ass wi h sh ubs (was e g ound)
Téglás-Új ehé ó EU249 H.b. Ma gins o maize  eld
Új ehé ó EU254 He . G ass (was e g ound)
*KNP IV-VI: Kiskunsagi Na ional Pa k, Regions IV-VI.
** I ish ype e-isola edin No . 1992 (Sjælland) o June 1994 (Kecskemé : EU333, EU335, EU347, EU348 and EU349).
*** Hb. Re-isola ed in June 1994 (EU339 nea Kecskemé ; EU369 a KNP IV; EU362 & EU367 a KNP V(1)).
he e ge (si e o EU167), whe e i was de ec ed in 5/20
samples, bu i was no isola ed in any o se en een sam-
ples om he pas u e (si e o EU176). This ime, H. bac-
e iopho a was p esen a bo h si es ( e ge: 1/20 samples;
pas u e: 2/17 samples), hough i had no been de ec ed a
ei he si e in he 1991 sampling p og amme.
The a e age numbe o bai insec s pa asi ised (lu-
minescen ) in he He e o habdi is-posi i e samples om
Kecskemé was simila o ha o Denma k and Es o-
nia, while signi can ly mo e insec s we e pa asi ised in
he Deb ecen samples (Fig. 3). To es whe he he di e -
en pa asi isa ion le els in he Deb ecen and Kecskemé
samples was due o di e ences in species composi ion
be ween he wo a eas, a compa ison was made es ing
only hose samples in which H. bac e iopho a was iden i-
 ed. H. bac e iopho a-posi i e samples om Kecskemé
yielded less han hal as many pa asi ised cada e s pe
sample as he H. bac e iopho a-posi i e Deb ecen sam-
ples (Kecskemé : x = 2.3±0.76, n = 6; Deb ecen:
x = 6.0±2.04, n = 4) and he di e ence was signi -
can a P= 0.086 (S uden ’s es ).
CROSS-BREEDING STUDIES
Hunga ian and Danish isola es which display an I ish
ype p o le a e in e e ile wi h each o he and wi h he
I ish ype isola e K122, om I eland (Table 4). When he
emales o he I ish ype isola e EU114 (Denma k) we e
c ossed wi h males o he NWE ype isola e EU85 (also
om Denma k), emb yonicle hal p ogeny esul ed (no in
able). C osses be ween Hunga ian isola es wi h he I ish
ype p o le and Hunga ian isola es wi h he HP88- ype
p o le ga e ise o emb yonic le hal p ogeny (da a no
shown).
Fig. 3. Mean numbe o luminescen Galle ia mellonella cada -
e s pe He e o habdi is-posi i e soil sample om Es onia, Den-
ma k (Sjælland), and Hunga y (Kecskemé and Deb ecen). Da a
a e o  s bai ing and o al ( s and second) bai ing. Ba s
accompanied by he same le e a e no signican ly di e en
(P <0.05, Duncan’s mul iple ange es ); lowe case le e s:
 s bai ing; uppe case le e s: o al bai ing.
Hunga ian isola es ha ing he same ITS egion MboI
es ic ion p o le as he H. bac e iopho a HP88 s ain
we e no in e e ile when c ossed wi h i . The c oss o
HP88 emales and Hunga ian HP88 ype males (EU362
and EU369) ga e s e ile F1 p ogeny while he ecip ocal
c oss esul ed in emb yonic le hals. C osses se up wi h
Da ms ad (a Eu opean isola e wi h he HP88 ype p o-
 le and in e e ile wi h HP88) ga e he same esul s wi h
hese isola es as did he c oss wi h HP88 i sel (Table 5).
None o he es ic ion enzymes used o diges ei he he
ITS egion o he NTS egion o he DNA gene complex
e ealed a di e ence be ween he Hunga ian HP88 ype
Vol. 1(3), 1999 327
C.T. G i  n e al.
Table 4. C osses be ween He e o habdi is isola es sha ing he “I ish ype” DNA ITS es ic ion p ole and esul ing in e ile
p ogeny (+).
Female Male
K122 EU94 EU106 EU333 EU349
(I eland) (Denma k) (Denma k) (Hunga y) (Hunga y)
K122 + + + + +
EU94 + + + + +
EU349 + + + + +
Table 5. C osses be ween isola es sha ing he H. bac e iopho aHP88 ype DNA ITS es ic ion p ole.
Female Male
HP88 Da ms ad EU362 EU369
(USA) (Ge many) (Hunga y) (Hunga y)
HP88 + + F1 F1
Da ms ad + + n.d. F1
EU369 e.l. e.l. + +
+ : c oss esul ed in e ile p ogeny.
e.l.: emb yonic le hal.
F1: s e ile F1 adul s.
n.d.: no de e mined.
isola es and HP88 i sel . Fig. 4 shows a HaeIII diges ion
p o le o he DNA NTS egion o  e Hunga ianH. bac-
e iopho a HP88 ype isola es. All o hese isola es sha e
he same NTS es ic ion p o le as he HP88 isola e, ha -
ing ou es ic ion agmen so ca 620, 332,197 and 148
bp. The ain bands isible on his gel mos p obably ep-
esen incomple ediges ion p oduc s.
Discussion
Isoelec ic ocusing elec opho eg amso soluble p o-
eins om IJ and he pa e n o he es ic ion enzyme di-
ges o he ITS egion o DNA e ealed ha bo h NWE
ype H. megidis and I ish ype He e o habdi is occu in
Denma k. Isola es showing he I ish ype PCR p o le
we e also eco e ed om soil samples collec ed in he
Kecskemé egion o Hunga y. The c oss-b eeding s ud-
ies showed ha hese Hunga ian and Danish I ish ype
isola es a e in e e ile wi h each o he and wi h he I ish
ype isola e K122 ( om I eland). This indica es ha he
I ish ypeo He e o habdi isis no es ic ed o he coas al
egion o I eland and B i ain (G i  n e al., 1994b; Ho-
minick e al., 1995), bu occu s in cen al and no he n
Eu ope as well.
Fig. 4. HaeIII es ic ion diges s o he PCR ampli ca ion p od-
uc s o he DNA non- anscibed space egion o Hunga ian
isola es o he HP88 (H. bac e iopho a) ype sepa a ed on a
2% aga ose gel. 1: HP88; 2: EU339; 3: EU362; 4: EU367; 5:
EU368; 6: EU369; M: 1 kb DNA ladde (Gibco BRL).
328 Nema ology
He e o habdi isspp. om Hunga y, Es onia and Denma k
NWE ype H. megidis p edomina es on he no he n
con inen al inge,being he only He e o habdi is isola ed
o da e om he Ne he lands (Hominick e al., 1995),
Belgium (Midu u i e al., 1996), no he n Ge many and
Poland (Smi s e al., 1991) and Es onia (p esen s udy).
The NWE ype has also been iden ied om he sou h
eas o England(Hominicke al., 1995) whe e he clima e
o B i ain mos esembles ha o he con inen al inge.
In con as , he I ish ype He e o habdi is is he only one
eco e ed in I eland and in all bu he sou h eas o G ea
B i ain. This migh sugges ha i is a species o mild, we
clima es. Howe e , i s disco e y a cen al Eu opean si es
in Hunga y and on a po ion o he con inen al inge in
Denma k, whe e i occu s wi h he NWE ype, sugges s
ha he pic u e is mo e complica ed: u he sampling
e o will clea ly be necessa y o delinea ei s dis ibu ion
ange. The appa en absence o he I ish ype in Es onia,
wi h i s ela i ely long cold win e s, would suppo he
hesis ha he I ish ype is a species o equi able clima es.
Mo eo e , bea ing in mind ha soils sampled we e o
a simila , sandy ex u e, i is s iking ha I ish ype He-
e o habdi is occu ed a a lowe p e alence owa d he
clima ically mo e ex eme cen e o he con inen (Hun-
ga y, 4% o si es) han in Denma k (23% o si es) o in
I eland and B i ain, whe e i was de ec ed a an a e age o
12% o si es (G i  n e al., 1994b). This sugges s ha i
is less success ul in he condi ionsp e ailing in Hunga y,
which would acco d wi h he poo e hea ole anceo I ish
ype isola es han o Hunga ian H. bac e iopho a epo ed
by Finnegan e al. (1999). The si e a which he I ish ype
was de ec edin bo ho he Hunga iansamplings(No em-
be , 1991 and June, 1994) was a oadside e ge shaded
by ees — i ually a small woodland; ew o he wooded
si es we e included in his s udy. In he Nege egion o
Is ael, He e o habdi is densi ies we e highe in plo s wi h
a high deg ee o shade (Glaze e al., 1996). Pe haps in
Hunga y, I ish ype He e o habdi is is a ou ed by wood-
land, whe e lowe empe a u es and highe soil mois u e
would be expec ed han in he neighbou ing g assland.
While he o he Hunga ian si e a which he I ish ype was
eco e ed was open g assland, i was bo de ed by eeds,
indica ing a high wa e able. The I ish ype was no e-
co e ed om his si e du ing he epea sampling in June
1994; howe e , a ha ime o he yea he nema odesmay
ha e been below he 10 cm soil dep h o which i was sam-
pled. In wa m egions, He e o habdi is ends o be ound
in he deepe soil laye s du ing he summe , especially
in soil which is mo e exposed o sola adia ion (Glaze
e al., 1996). The I ish ype isola es om Denma k and
Hunga y ha e no ye been ully cha ac e ised o biolog-
ical ai s; gi en he di e si y amongs isola es o he ype
om B i ain and I eland (Hass, 1996) i would no be su -
p ising i locally adap ed eco ypeswe e de ec edamongs
hem.
A numbe o He e o habdi is isola es collec ed in Hun-
ga y show DNA ITS and DNA NTS DNA es ic ion
p o les iden ical o ha o he Ame ican isola eo H. bac-
e iopho a, HP88, using key diagnos ic es ic ion en-
zymes. Howe e , he Hunga ian isola es a e no in e e -
ile wi h HP88. HP88-compa ible nema odes do occu in
Eu ope, howe e ,as e idencedby he c oss-b eedingsuc-
cess wi h he Da ms ad s ain. The incompa ibili y be-
ween HP88 and he Hunga ianHP88-like isola esis a he
pos -ma ing le el, and esul s in ei he hyb id in iabili y
o hyb id s e ili y dependingon he di ec iono he c oss.
Emb yos we e o med in all o he c osses ha we e ca -
ied ou . These emb yos g ew in o appa en ly no mal bu
s e ile adul si he ma e nal pa ne o he c oss was om
ei he he HP88 o Da ms ad s ain. Whe he gene ic o
cy oplasmicma e nal ac o s play an impo an ole in he
ep oduc i eisola ion be ween hese isola es has no been
de e mined.
The esul s o his su ey p o ide u he e idence o a
geog aphicsepa a ion in Eu ope be ween he H. megidis-
like nema odes ( he I ish and NWE ypes o He e o-
habdi is), ound p edominan ly in no he n Eu ope, and
he H. bac e iopho a g oup ound in sou he n and cen-
al Eu ope. Howe e , he occu ence o he I ish g oup
in Hunga y (p esen s udy) and o H. megidis in G eece
(Men i e al., 1997) clea ly indica es ha a di ision be-
ween a no he n ‘megidis‘ g oup and a sou he n H. bac-
e iopho a g oup is oo simplis ic.
The eco e y a e o He e o habdi is was much highe
in his s udy, in which si es wi h sandy soils we e speci -
ically a ge ed o sampling, han in non- a ge ed su eys
conduc ed in neighbou ing a eas o Eu ope. Thus, He e-
o habdi is was eco e ed a one hi d o si es in Hun-
ga y, bu was no eco e ed in he su ey o Czechoslo-
akia (M ácek, 1980). Simila ly, he eco e y a e o He-
e o habdi is a 38.5% o si es in Sjælland is conside -
ably highe han in Wes -Flande s (Belgium) (5% o si es)
(Midu u ie al., 1996)o in he Ne he lands(13% o si es)
(Hominick e al., 1995). The e  cacy o a ge ed sam-
pling in eco e ing s ains o He e o habdi is was p e i-
ously demons a ed in I eland and B i ain: G i  n e al.
(1994b) eco e ed He e o habdi is om 4-45% o sandy
si es, while in gene al su eys, i ei he was no de ec ed
(Blackshaw, 1988; Boag e al., 1992; Gwynn & Richa d-
Vol. 1(3), 1999 329