Isolation and characterisation of Heterorhabditis spp. (Nematoda: Heterorhabditidae) from Hungary, Estonia and Denmark
Abstract
Targeted surveys were conducted for the entomopathogenic nematode Heterorhabditis in areas of Denmark, Estonia and Hungary. Isolateswere identiŽ ed by IEF, PCR and cross-fertilitytests as belonging to three distinct taxonomic groups: H. bacteriophora, the north-west European (NWE) type of H. megidis and the Irish type of Heterorhabditis. The Irish and NWE types of Heterorhabditis were both present in Denmark (at six and four sites, respectively),while only the NWE type was recovered in Estonia. H. bacteriophora was the dominant heterorhabditididentiŽ ed in Hungary (ten sites), but the Irish typewas also detected at two sites. This is the Ž rst report of the Irish type of Heterorhabditis on continental Europe. Co-occurrence of two Heterorhabditis types at a single site was noted in Denmark (Irish and NWE) and in Hungary (Irish and H. bacteriophora). Heterorhabditiswas recovered at 38.5% of sites (n = 26) in Denmark (north coast of Sjælland), 27.3% of the coastal sites (n = 22) in Estonia, and 32.6% of sites (n = 46) in Hungary.
Full text
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 ica 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 ied 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 conspecic 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 ied 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 ica 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 ied 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 ied 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 signicance 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 ied 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 signican 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 ole 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 ole.
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 ied 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