Folia Microbiol 46 (5), 423-426 (2001) http: //www .biomed. cas. cz/mbu/folia/
Spacer Region of rDNA of the Truffle Terfezia
terfezioides in Europe
G.M. KOVACS a*, S. RUDNOY b, C. V,~GVOLGYI c, D. L,/~SZTITY b, I. RACZ b, Z. BRATEK b
aDepartment of Botany. UniversiO, of Szeged. 6701 Szeged, Hungary
lax +36 1 467 4076
bDepartment of Plant PIo,siolog),. EOtvds Lordnd UniversiO,. 1445 Budapest. Hungary
CDepartment of Microbiology. Umverstty of Szeged. 6701 Szeged. Hungary
Invariability of the Internal Transcribed
Received 12 Aprd 2001
ABSTRACT. ITS regions (internal transcribed spacers - 1225"1
and ITS2 - with the 58S gent of the nuclear rDNA) of 25 fruit
body samples of Terfezia terfe-ioides, originating front Hungary
and Italy, were compared. The amplification and sequencing of
the ITS region was successful with both the ITSI-ITS4 and
/TSIF-ITS4 primer pairs. No differences of the restriction frag-
ment length polymorphism profiles were detected among 19
samples collected in one place at the same time. The sequences
of tire ITS region o19 samples collected m different localities
were highl)invariable, differing m only two bases. Thus the
mtraspecific honrogeneity of the ITS region seems to be an mrpor-
tant species-specific characteristic of 22 terfezioides m contrast
to other Terfezia species. As the samples of the species were
collected from different and distant localities of Europe, the ITS
sequence of 22 terfezioides can be considered a very conserva-
tive, reliable molecular marker of the fungus.
Terfezia terfezioides (MATT.) TRAPPE (Ascomycetes, Pezizales, Terfeziaceae) is a hypogeous fun-
gus, which has been reported several times from different countries and regions of Europe, and from other
parts of the world since its first description in Italy (Mattirolo 1887; Babos 1981; Kir~ily et al. t992; Zhang
1992; Montecchi and Lazzari 1993; Lawrynowicz et aL 1997). It was mainly found in Hungary and in North-
Serbia - both areas are in the Eupannonicum flora region - naturalized in mixed black locust forests on sandy
soils in the catchment area of the Danube river (Babos 1981; Kir~.ly et al. 1992; Lawrynowicz et aL 1997).
The ecology and mycorrhizal characteristics of other Terfezia species were discussed in several pre-
vious works (Dexheimer et al. 1985: Kirfily et al. 1992; Taylor et al. 1995: Bratek et al. 1996); only some of
the species were studied by molecular methods (Pacioni et al. 1997: Kagan-Zur et al 1998, 1999: Aviram et
al. 2000a,b). Although the intrageneric relationships in the TerJezia genus were studied by Diez et al. (1998)
and T terfezioides itself was also included in the molecular systematical studies of the Pezizales (Percudani
et al. 1999), the available molecular-biological taxonomical data on this species are insufficient.
The internal transcribed spacer region (ITS) containing the 5.8S gene of the nuclear rDNA has been
reported to be highly conserved within various species showing inter- rather than intraspecific variability
(White et al. 1990; Gardes et al. 1991; Gardes and Bruns 1993). Still, heterogeneity of the ITS region was
found on intraspecific level in several fungi (Henrion et al. 1992, 1994; K~r6n et aL 1997; Gottlieb and Lich-
wardt 2001), including the ascomycetous Tuber (Gandebouf et al. 1997; Paolocci et al. 1997), and TerJezia
genus (Kagan-Zur et al. 1999). The specificity of the ITS-region was checked by Bruns and Gardes (1993) to
ensure its successful use in molecular probes.
Here we describe the variability of the ITS region of T. terfezioides, intraspecific ITS heterogeneity
studied by restriction fragment length polymorphism analysis (RFLP) and by comparison of the sequences.
MATERIALS AND METHODS
Samples. Twenty-five samples were collected from 6 different locations (5 from Hungary and 1 from
Italy) (Table I). The heterogeneity within one habitat was determined by RFLP analysis in 19 fruit bodies
collected in Kunfeh4rt6 (Hungary) at the same time.
DNA isolation. The DNA extraction was carried out according to Gardes et aL (1991) with slight
modifications. Small amount of dried vegetative mycelium or small parts of the desiccated fruit bodies
(Table I) were thoroughly ground in liquid nitrogen, resuspended in lysis buffer (2 % hexadecyltrimethyl-
ammonium bromide, CTAB; 20 mmol/L EDTA, pH 8; 100 mmol/L Tris-HCl, pH 9; 1.4 mmol/L NaCI) or
* Present address: ~'eterinary Medical Research lnstmae, Hungarian Academy of Sciences. 1581 Budapest, Hungary
424 G.M KovAcs etal
ground with micropestles and sand in Eppendorftubes in the same buffer. After incubation (65 ~
samples were extracted twice with an equal volume of chloroform and centrifuged (12 000 g, 15 min) after
both extractions. The DNA was precipitated with two volumes of ethanol; after at least l h (at -20 ~
pelleted by centrifugation (12 000 g, 30 rain). The pellet was washed twice with 70 % ethanol, dried and
redissolved in 70 gL sterile Milli-Q water (Millipore).
I h), the
Table i. The examined Terfezia terfezioides material
GeneBank TSeq c
of material a
2. 12. 1995
30 08 1999
I 0.09. 1991
13. 11. 1996
15. 10. 1998
KMG 10124 e
ae - desiccated fruit bodies: m - mycelial culture isolated from ffuitbodies.
bBratek - collection of Z. Bratek, KMG - collection ofG.M Kov~ics.
dTseq - sequence types: see Results and Discussion.
dSampled in Italy: other samples from Hungary.
eOrigmatmg from collection specimens of Percudani et aL 1999.
DNA amplification. In case of samples 5-9, PCR amplification of the ITS region was carried out
with the primers ITSI and ITS4 according to Henrion et al. (1992) with some modification. The reaction
mixture contained 0.1 volume 10x PCR buffer (Zenon), 200 p, mol/L each of dATP, dCTP, dGTP and dTTP
(Pharmacia), 2.5 mmol/L MgCI2, 0.5 lamol/L of each primer, 0.5 U Taq DNA polymerase (Zenon) and
5 ng/gL of genomic DNA extract. The amplifications were performed with Perkin Elmer PCR System 2400,
programmed for a denaturation step (93 ~ 3 min), followed by 35 cycles of denaturation (95 ~
annealing (55 ~ 2 min) and extension (72 ~ 2 rain); the thermal cycling was ended by a final extension at
72 ~ for 10 min. The amplification of the ITS region of the remaining samples was done with ITSIF and
1TS4 primers according to Gardes and Bruns (1993); the reaction conditions were the same as above but the
samples were overlaid with sterile mineral oil (Sigma), and PTC-100 DNA thermocycler (MY Research) was
Digestion. The RFLP analysis of the ITS region of the 19 samples collected from Kunfehdrt6 was
made using Cfol, EcoRl, Hinfl and Xhol restriction enzymes (Kramer Biotech) following manufacturer's
directions. Digestion products were analyzed by electrophoresis on 1.7 % agarose gel in TAE buffer
(40 mmol/L Tris-acetic acid, pH 7.6; 1 mmol/L Na2EDTA) containing 0.5 mg/L ethidium bromide.
Sequencing. For direct sequencing, PCR products were purified with Prep-A-Gene matrix (BioRad).
ABi PRISM BigDye Terminator Kit (Perkin Elmer) was used for cycle sequencing and the electrophoresis
was carried out on ABI PR1SM 310 Genetic Analyzer according to the manufacturer's instructions. The sequen-
cing was carried out with the primers used for the amplification.
RESULTS AND DISCUSSION
The PCR amplification of the ITS has been commonly used in the molecular studies of fungi since
the design of primers for this purpose (White et al. 1990: Gardes and Bruns 199.3; Bruns et al. 1998). This
amplification could sonnetimes not be performed, e.g., in Tuber aestivum VITT. and Tuber uncinatum CH.
truffle species (Henrion et al. 1994), although the ITS of the former species had been amplified (Lanfranco
et al. 1995). In the case of 7~ terfezioides the amplification and also the sequencing of the ITS region were
successful with both primer pairs ITSI-ITS4 and ITS1F-ITS4. The reproducible amplification of the ITS
with the latter, fungal-specific primer pair brings valuable information for mycorrhizal studies of the truffle
because it makes it possible to amplify the fungal ITS from the mixed DNA extracted from mycorrhizae
2001 INTERNAL TRANSCRIBED SPACERS REGIONS OF rDNA OF T terfeztotdes 425
(Gardes and Bruns 1993). Thus the separated amplification of plant ITS, which is necessary with non-spe-
cific primers (Lanfranco 1995; Kagan-Zur et al. 1999), could be avoided.
The ITS region of the 19 fruit bodies collected from one locality showed invariability in their RFLP
patterns tested with the 4 enzymes. EcoRI did not digest the amplified region (approximately 740 bp) while
the digestion with CfoL Hinfl and XhoI resulted in 2 (approximately 400 and 300 bp), 3 (approximately 340,
190 and 145 bp) and 2 (approximately 555 and 185 bp) detectable bands. The restrictions checked after
the sequencing and the size of the bands - considering the undetectable small fragments and the unsequenced
but amplified regions - were acceptable. The intraspecific variability in RFLPs of the ITS region reported
previously (Henrion et al. 1992; Kg_r6n el al. 1997; Paolocci et al. 1997) has usually been found among dis-
tant locations like in Laccaria species (where the differences correlated with the geographical distances;
Henrion et aL 1992), in ectomycorrhizal fungal species of Fennoscandia (K~_r6n et aL 1997) and in Tuber
indicum COOKE et MASSE (Paolocci el al. 1997). TerJeziapfeilii HENN. showed two different ITS RFLP
profiles from the same locality and even a mixture of two RFLP types within the vegetative mycelium
isolated from one fruit body (Kagan-Zur et al. 1999). To check the variability on the sequence level three
samples have been chosen and sequenced to represent the location.
Nine sequences of the ITSI and ITS2 spacers and the 5.8S rRNA gene of samples collected at 6 dif-
ferent locations showed high homogeneity. Two sequence types were found (marked as type A and B); these
differed only in two nucleotides, one in the ITS1 and one in the 1TS2 region. Five samples have the B type
and 4 have the A type sequence (Table 1). The sequences were the same within the same (e.g., samples 2-4)
or close localities (e.g., samples 5-7) similarly to the samples collected at distant locations (e.g., samples 1, 2
and 8); the two sequence types seemed not to be separated geographically (samples 5-9).
This high invariability of the ITS region is an important species-specific characteristic of T. terJezi-
oides in contrast to other Terfezia species studied previously (Kagan-Zur et al. 1998, 1999; Aviram et al.
2000a,b). In the case of Terfezia boudieri CHATIN, intraspecific variation of the ITS sequence has been
detected among 3 strains of the species (Kagan-Zur et aL 1998) and two different ITS forms within one
hypha (Aviram et aL 2000b). Intraspecific variability has been also detected in the case of T. pfeilii HENN.
with the above detailed ITS RFLP profiles within one locality (Kagan-Zur et al. 1999) and the sequences of
the ITS have also shown differences (Aviram et aL 2000a).
The ITS sequence of T. terfezioides can be considered a firm, representative molecular marker of
this species in its whole European area, where the most reliable data have been obtained. This could help in
checking this fungus reported in China (where probably a misidentification of 7~ terJezioides occurred and
the data are difficuh to use; Zhang 1992) and could also give valuable help in the study of the North-Ame-
rican hypothesized sister species of the fungus.
Species-specific invariability of the ITS region is important in the studies of mycorrhizal connect-
ions of this fungus. Robinia pseudoacacia L. (Kira.ly et al. 1992: Bratek et al. 1996) and some other plants
have been assumed to be a mycorrhizal partner of this truffle (Kir~ily et aL 1992). Based on our study not just
the successful amplification with fungal specific primers and the RFLP profiles could help in finding the
natural mycorrhizal partners of T. terfezioides, but such a conservative genetic marker makes possible the
design of species-specific PCR primers as in the case of ascomycetous Tuber melanosporum VITT., T. bru-
male VITT. and Z indicum (Rubini et al. 1998) and T. borchii VlTT. (Bertini et al. 1998) and in basidio-
mycetous Tricholoma matsutake (S. ITO et IMAI) SING. (Kikuchi et al. 2000).
This work was supported in part by grants OMFB05695960417 (National Committee for Technologtcal Development:
OMFB). and OTKA T032738 (Hungarian Scientific Research Fund; OTKA). Authors are grateful to R. Percudani and A. Zambonelli
tbr the herbarial material, thank E. Jakucs for her valuable advice and B.L. Rosenberg for his help; authors also thank Kiskuns/tg
National Park tbr the permission to collect samples in the protected area of the Park.
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