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Fungal Diversity
51
Morphological and molecular characterisation of mycelia of
ectomycorrhizal fungi in pure culture
Mirco Iotti1, Elena Barbieri2, Vilberto Stocchi2 and Alessandra
Zambonelli1*
1Dipartimento di Protezione e Valorizzazione Agroalimentare, Università degli Studi di
Bologna, via Fanin 46, 40127 Bologna, Italy
2Istituto di Chimica Biologica “Giorgio Fornaini”, Università degli Studi di Urbino, 61029
Urbino (PU), Italy
Iotti, M., Barbieri, E., Stocchi, V. and Zambonelli, A. (2005). Morphological and molecular
characterisation of mycelia of ectomycorrhizal fungi in pure culture. Fungal Diversity 19: 51-
68.
Boletus edulis, Boletus aestivalis, Boletus luridus, Amanita muscaria and Hebeloma radicosum
mycelia were isolated in pure culture and characterised by morphological and molecular
methods. Molecular identification was performed by sequence analyses from the ITS region of
nuclear ribosomal RNA genes. The phylogenetic affiliation of the isolated mycelia were
evaluated by comparison of their ITS sequences with those deposited in the GenBank database.
Pure cultures of isolates of the different fungal genera under investigation showed differences in
growth rate, colony morphology and/or hyphal biometric characters. In contrast, the
morphological characteristics of the mycelia of the three Boletus species were similar, but these
species were distinguished by ITS data. Problems remain, however, in the affiliation of these
ITS sequences with those of the B. edulis group that are currently deposited in public databases.
Key words: Amanita muscaria, Boletus aestivalis, Boletus edulis, Boletus luridus, Hebeloma
radicosum, molecular characterisation, morphological characterisation, pure culture.
Introduction
Mycorrhizal fungi are of great interest for environmental and forestry
application due to the advantages that mycorrhizae provide for the host plant.
Furthermore, some ectomycorrhizal fungi are also of economic importance in
that they are edible, such as truffles, boletes and chanterelles (Watling, 1997;
Tibiletti and Zambonelli, 2000). Several species are used in the commercial
production of infected plants and the starting point is generally seedling
inoculation either with spores or mycelial cultures (Hall and Wang, 1998). The
first step in the production of infected plants using pure cultures of mycorrhizal
*Corresponding author: A. Zambonelli; e-mail: zambonel@agrsci.unibo.it
52
fungi is to obtain axenic-mass cultures under controlled conditions (Kuek,
1994). Even when the fruit bodies used to obtain these pure cultures are
unambiguously identified based on their morphological characters, the identity
of the isolated mycelia should also be confirmed by other methods (Iotti et al.,
2002). Misidentification of pure cultures can occur, in particular for those
fungal isolates that grow very slowly on synthetic media, such as
ectomycorrhizal fungi (Mello et al., 2001; Bridge et al., 2003). Some
morphological characters of colonies and hyphae have been used to confirm the
identity of ectomycorrhizal fungi in pure culture (Brundrett et al., 1996) but
these characters are not very distinctive with respect to those of sporulating
fungi (Arx, 1980; Gravesen et al., 1994). Recently, new molecular methods
primarily based on the polymerase chain reaction (PCR) have provided rapid,
sensitive, and reliable alternatives for identifying ectomycorrhizal fungi at any
phase of their life cycle (Amicucci et al., 2001; Horton and Bruns, 2001).
Despite their considerable value, molecular methods have so far been
applied for the identification of pure cultures for only a restricted number of
ectomycorrhizal fungi, primarily to those of Tuber spp. (Rossi et al., 1999; Iotti
et al., 2002). The aim of this study was, therefore, to extend the molecular and
morphological characterization of ectomycorrhizal mycelia to basidiomycetous
species of ecological or economic importance.
Materials and methods
Isolation of the mycelia
Basidiome of Amanita muscaria (L.) Hook., Boletus edulis Bull., B.
aestivalis (Paulet) Fr. (= B. reticulatus Schaeff.), B. luridus Schaeff. and
Hebeloma radicosum (Bull.) Ricken were collected in summer-autumn 2000-
2001 and August 2002 (B. luridus, herbarium number 1968) in three different
sites of the Emilia Romagna Apennines (Italy) (Table 1), and dried specimens
of each species were deposited in the herbarium of the “Centro di Micologia” of
Bologna. The species were identified by morphological methods, according to
Breitenbach and Kränzlin (1995).
Blocks of tissue 1-2 mm across, aseptically excised from the inner part of
the cap, were cultured on modified Woody Plant Medium (mWPM, Table 2)
(Lloyd and McCown, 1980) and incubated in the dark, at 22 ± 1°C, for 2-3
months before the first transplanting. The isolated mycelia were then
maintained in culture in half strength Potato Dextrose Agar (20 g/l) (hsPDA)
(Difco, Detroit, MI, USA).
Fungal Diversity
53
Table 1. Provenance, herbarium number (herbarium of the “Centro di Micologia” of Bologna,
Italy) and probable host plant of the isolated ectomycorrhizal fungi.
Species Herb.
n.
Collection locality Probable host plant Date of
isolation
Strain
Boletus aestivalis
Boletus edulis
Boletus luridus
Boletus luridus
Amanita muscaria
Hebeloma radicosum
1773
1640
1807
1968
1642
1647
Loiano
Castiglione dei Pepoli
Zocca
Zocca
Castiglione dei Pepoli
Castiglione dei Pepoli
Castanea sativa Mill.
Fagus silvatica L.
Castanea sativa Mill.
Castanea sativa Mill.
Fagus silvatica L.
Fagus silvatica L.
15-06-01
20-10-00
10-08-01
08-08-02
20-10-00
20-10-00
Bre1
Edu2
Blu3
-
Amu1
Hra1
Table 2. Composition of mWPM.
Compound Concentration Compound Concentration
MgSO4•7H2O
KH2PO4
CaCl2•2H2O
NH4NO3
Ca(NO3)2•4H2O
K2SO4
MnSO4•H2O
ZnSO4•7H2O
0.37 g/l
0.17 g/l
0.096 g/l
0.4 g/l
0.556 g/l
0.9 g/l
22.3 mg/l
8.6 mg/l
H3BO3
Na2MoO4•2H2O
CuSO4•5H2O
FeSO4•7H2O
NaEDTA•2H2O
Myo-inositol
D(+)-glucose
Agar
6.2 mg/l
0.25 mg/l
0.025 mg/l
27.8 mg/l
37.3 mg/l
0.1 g/l
8 g/l
10 g/l
Morphological characterization
The morphology of the colonies was observed both on mWPM and
hsPDA plates and described according to Stalpers (1978). Petri dishes of 5 cm
diam. containing 12 ml of mWPM were inoculated with a 0.7 cm disk of
mycelial felt taken from the rim of 30 day-old cultures on hsPDA. Plates were
kept wrapped in Parafilm “M” (American National CanTM, Chicago, IL,
USA) to avoid dehydration of the culture media and maintained in the dark, at
22 ± 1°C. The growth of the fungal colonies was recorded every week along
two preset diametrical lines. There were five replicate plates in each treatment
and the whole experiment was repeated three times.
Micro-morphological features of the cultures were only examined in
mWPM since in this medium the fungal colonies are looser than in hsPDA,
making microscopic analyses easier. Agar plugs (1 cm2 × 0.3 cm high)
collected from the surface of the 60 day-old fungal colonies were examined
under a light microscope Laborlux 12 (Leitz, Wetzlar, Germany); images were
captured using a high-resolution colour video camera (JVC, Yokohama, Japan)
and 50 measurements per parameter made with the Axio Vision 2.05 image
analysis software (Zeiss, Jena, Germany). The hyphal growth unit (total hyphal
length / number of hyphal tips) (Trinci, 1973), branching angle, hyphal
54
diameter, septal distance and percentage of clamps were measured in the
peripheral growth zone of the mycelium.
Molecular characterisation
Molecular identification of the isolated mycelia and of the Boletus luridus
1968 basidiome was performed using sequence data of the ITS region of the
nuclear ribosomal DNA. Total genomic DNA was isolated from 100 µg of
fungal tissue (one-month-old mycelial cultures or fresh basidiome) by DNeasy
Plant Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's
instructions and then eluted in 50 µl of sterile water. ITS-1, 5.8S and ITS-2
regions were amplified in a 50 µl volume reaction containing 1-10 ng of
genomic DNA, using the primers pair ITS1 and ITS4 (White et al., 1990) in a T
gradient Thermal Cycler (BIOMETRA, Göttingen, Germany) according to
Amicucci et al. (1996). PCRs were performed using 2.5 units of Taq DNA
polymerase (Fermentas, Vilnius, Lithuania).
The amplified products were purified by Gene Clean II kit (BIO 101,
Vista, CA, USA) and sequenced using the two primers mentioned above.
Sequence reactions were run in a ABI PRISM 3700 DNA Analyzer (Applied
Biosystem, Foster City, CA, USA) with Big Dye Terminator v3.1 chemistry.
The ITS sequences of the different species were compared to those available in
the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) using the
BLASTN search (Altschul et al., 1997).
Sequences were aligned using PileUp program in the Genetics Computer
Group (GCG) Package version 9.1, Madison, Wisconsin. The programs utilised
for the phylogenetic analyses of the complete ITS1-5.8S-ITS2 region are
included in the PHYLIP software package version 3.5c (Felsenstein, 1993).
Distance matrix was inferred using the DNADIST program and the
phylogenetic trees were constructed from the evolutionary distance matrix with
FITCH and using the DNAML and DNAPAR programs for the maximum
likelihood and the parsimony analyses, respectively. The robustness of the tree
topologies was evaluated by bootstrap analysis based on 200 re-samplings of
the sequence alignment. Bootstraps were performed with DNABOOT and tree
branches with a confidence level > 75% were considered significantly robust.
The TreeView program was used to plot the treefiles (Page, 1996).
Nucleotide sequence accession numbers
The ITS sequences obtained in this study have been deposited in
GenBank with the following accession numbers: Amanita muscaria mycelium
(Amu1 strain) (AY278768), Boletus luridus mycelium (Blu3 strain)
Fungal Diversity
55
(AY278765), B. luridus fruit body (AY278766), B. aestivalis mycelium (Bre1
strain) (AY278769), B. edulis mycelium (Edu2 strain) (AY278764), Hebeloma
radicosum mycelium (Hra1 strain) (AY278767). The other sequences included
in this study were obtained from the GenBank database (Table 3).
Results
Morphological and growth characteristics
The growth rate of mycelia on mWPM is reported in Fig. 1. During the
exponential growth phase, the radial growth of the mycelium of Hebeloma
radicosum was about twice as high as that in other species. The Amanita
muscaria mycelium showed the shortest lag phase. The stationary phase did not
occur for Boletus mycelia within eight weeks of analysis.
The macroscopic characters of mycelial cultures and the biometric
characteristics of the hyphae and clamp frequency of the isolated mycelia are
reported in tables 4 and 5, respectively.
Amanita muscaria colonies were white and the mycelium developed only
at the surface of the media on both mWPM and hsPDA. They did not exude
pigments or droplets. The hyphae often grew interlaced (Fig. 2b) and formed
frequent anastomoses. Clamp connections were short, large, and slightly curved
(Fig. 2d).
Hebeloma radicosum colonies were cream with abundant aerial mycelium
on mWPM, and yellowish with crustose brown areas on hsPDA. They did not
exude pigments or droplets. The hyphae only rarely formed anastomoses.
Clamp connections were short, large, and abrupt (Fig. 2e).
Boletus edulis colonies were white, whereas those of B. aestivalis and B.
luridus were cream and pale-brown, respectively, on both tested media. On
hsPDA, mycelia of the three Boletus species exuded brown pigments, that
coloured the medium from yellowish to brownish. Moreover, on that medium,
Boletus colonies exuded pigmented droplets from the aerial hyphae. Conversely
they did not secrete pigments and droplets in the mWPM. The mycelium of all
the studied Boletus species developed both on the surface of the media and
within them but only the hyphal morphology of B. luridus was greatly modified
within the medium (Fig. 2a) showing a helical growth as already reported by
Iotti et al. (2002) for Tuber mycelia. Hyphal swelling was common in the three
Boletus species (Figs. 2c-f). Hyphal septa were generally localised close to the
hyphal branches and lacked clamp connections.
56
Table 3. Species analysed in this study.
Species GenBank
accession no.
Lenght
(bp)
References
A. muscaria (L.) Hook. AF085490 628 Lim and Jung (1998)
AB081295 695 Oda
et al. (2002)
Z54294 669 Nehls (1995) *
AJ549964 634 Schmid et al. (2003) *
A. rubescens (Pers.) Gray AF085484 643 Lim and Jung (1998)
AB015682 723 Oda et al. (1999)
A. spissa (Fr.) P. Kumm. AF085486 653 Lim and Jung (1998)
A. citrina (Schaeff.) Pers. AB015679 734 Oda et al. (1999)
AF085489 660 Lim and Jung (1998)
AY325846 506 Hallen et al. (2003) *
A. phalloides Fr. AY325836 486 Hallen et al. (2003) *
AJ308097 697 Vasilenko and Rtischeva
(2001) *
A. virosa (Fr.) Bertill. AY325829 480 Hallen et al. (2003) *
AB015676 695 Oda
et al. (1999)
A. caesarea (Scop.) Pers. AY486237 515 Bernedo Cornejo et al.
(2003)*
A. gemmata (Fr.) Gillet AF335440 1217 Berbee et al. (2001) *
A. pantherina (DC.) Krombh. AB080978 704 Oda et al. (2002)
AB015701 712 Oda et al. (1999)
H. radicosum (Bull.) Ricken AF124700 610 Aanen
et al. (2000)
H. truncatum (Scaeff.) P. Kumm. AF124701 616 Aanen et al. (2000)
H. sinapizans (Fr.) Sacc. AF124682 614 Aanen et al. (2000)
AY320380 660 Boyle et al. (2003) *
H. mesophaeum (Pers.) Fr. AY311521 662 Boyle et al. (2003) *
AF124692 615 Aanen et al. (2000)
H. senescens (Batsch) Berk. & Broome AY312987 663 Boyle et al. (2003) *
H. edurum Métrod AF124698 614 Aanen et al. (2000)
H. sarcophyllum (Peck) Sacc. AF124715 610 Aanen et al. (2000)
H. sacchariolens Quél. AY312985 663 Boyle
et al. (2003) *
AF124689 612 Aanen et al. (2000)
H. pallidoluctuosum Gröger &
Zschiesch.
AY311526 664 Boyle et al. (2003) *
H. pusillum J.E. Lange AY312982 659 Boyle
et al. (2003) *
AF124702 609 Aanen
et al. (2000)
H. helodes J. Favre AY311516 659 Boyle
et al. (2003) *
AF124710 606 Aanen et al. (2000)
H. crustuliniforme (Bull.) Quél. AF124708 610 Aanen
et al. (2000)
B. luridus Scaeff. AJ419191 644 Martin and Raidl (2002)
Uncultured ectomycorriza AF465183 648 Selosse et al. (2002)
B. rhodoxanthus (Krombh.) Kallenb AJ419189 732 Martin and Raidl (2002)
B. calopus Pers. AJ296293 680 Martin and Raidl (2002)
B. fragrans Vittad. AJ419186 686 Martin and Raidl (2002)
B. impolitus Fr. AJ419187 653 Martin and Raidl (2002)
Fungal Diversity
57
Table 3 continued. Species analysed in this study.
Species GenBank
accession no.
Lenght
(bp)
References
B. erythropus Fr. AJ496595 602 Martin and Raidl (2002)
B. aestivalis (Paulet) Fr. AY130295 957 den Bakker
et al. (2002) *
B. pinicola (Vittad.) A. Venturi AJ419190 642 Martin and Raidl (2002)
B. edulis Bull. AJ419182 539 Martin and Raidl (2002)
AF074921 601 Grubisha
et al. (2002)
AJ416955 218 Moor et al. (2002)
Agrocybe praecox (Pers.) Fayod AF124713 611 Aanen et al. (2000)
Pisolithus tinctorius (Pers.) Coker &
Couch
AF374710 640 Martin
et al. (2002)
* published only in GenBank database.
Table 4. Macroscopic characters of mycelial cultures.
Strain Characters of the mat
Advancing zone Aerial mycelium Colony colour
Mycelial growth Outline Texture of mat
Reverse
mWPM hsPDA mWPM hsPDA mWPM hsPDA mWPM hsPDA mWPM hsPDA
Amu1 appressed appressed even even felty felty white white slightly
darkened
slightly
darkened
Hra1 appressed submerged bayed fringed felty subfelty cream yellowish unchanged unchanged
Edu2 appressed appressed bayed bayed felty felty white white darkened darkened
Bre1 appressed appressed fringed fringed felty felty cream cream darkened darkened
Blu3 appressed submerged even even felty felty pale brown pale brown darkened darkened
Table 5. Hyphal morphological characteristics of the isolates.
Strain Hyphal growth
unit (µm)
Branching angle
(°)
Septal distance
(µm)
Hyphal
diameter (µm)
Clamp
frequency
(%)
Bre1
Edu2
Blu3
Amu1
Hra1
427.90 ± 159
(177.26-947.41)
473.14 ± 168.12
(201.56-1054.4)
466.91 ± 165.01
(259.8-979.17)
462.20 ± 206.12
(216.86-1102)
426.75 ± 256.11
(122.88-985.23)
54.76 ± 14.35
(33.45-81.54)
32.40 ± 10.57
(20.71-64.61)
40.72 ± 10.72
(27.43-70.77)
59.52 ± 13.46
(36.42-93.12)
58.67 ± 17.09
(31.42-89.55)
78.82 ± 20.94
(35.37-130.99)
82.71 ± 27.84
(36.67-130)
85.25 ± 25.30
(30.93-133.39)
40.15 ± 14.07
(20-63.22)
46.19 ± 13.38
(20.92-78.37)
2.47 ± 0.23
(2.04-2.98)
2.75 ± 0.27
(2.37-3.4)
2.35 ± 0.24
(1.85-2.84)
1.99 ± 0.21
(1.62-2.37)
2.22 ± 0.25
(1.74-2.78)
0
0
0
24.72
62.50
Data are the mean of 50 measures from three different Petri dishes.
58
Molecular characterisation
Comparison of the ITS sequences obtained from the isolated mycelia with
the sequences available in the GenBank databases allowed us to analyse the
phylogenetic affiliation of Amanita muscaria, Boletus luridus, B. aestivalis, B.
edulis, and Hebeloma radicosum.
ITS sequence of Blu3 strain, isolated from a fruit body of Boletus luridus,
showed the highest levels of similarity (99%, identity = 598/601 nt) with a
nearly complete ITS1-5.8S-ITS2 sequence of an uncultured ectomycorrhiza of
presumptive Boletaceae family (accession number AF465183), described by
Selosse et al. (2002), and with our sequence of the B. luridus basidiome
(herbarium number 1968) collected in Italy; the similarity with the sequence of
a strain labelled B. luridus (accession number AJ419191) by Martin and Raidl
(2002) was only 94% (identity = 579/613). The phylogenetic position shown in
Fig. 3 confirms that our Blu3 strain corresponds to B. luridus.
The molecular data available for B. edulis are confusing and the
molecular identification of both B. edulis and B. aestivalis is contradictory.
Several sequences with a significantly high level of ITS similarity (> 90%)
from B. edulis and B. aestivalis mycelia were partial sequences containing only
the ITS-1 region of about 200 nt.
In this study, the phylogenetic analysis was carried out using only nearly
full length ITS1-5.8S-ITS2 sequence available in the databases; however to
better analyse the molecular data existing, Fig. 4 shows a pairwise alignment
based on the best available sequences of the ITS-1 region of B. edulis, in which
it is possible to verify the high level of similarity (identity = 161/161 nt) of our
B. aestivalis sequence with the ITS-1 of a B. edulis sample (accession number
AJ416955) described as "Chinese king bolete" by Moor et al. (2002).The
phylogenetic position of the nearly complete ITS1-5.8S-ITS2 sequence from B.
edulis mycelium shows a monophyletic origin with respect to the other edible
boletes but branches separately from the B. edulis described by Grubisha et al.
(2002) (accession number AF074921) and Martin and Raidl (2002) (accession
number AJ419182) (Fig. 3). The ITS sequence obtained from our B. edulis
mycelium showed 99% similarity (identity = 717/719 nt) with a ITS sequences
of B. aestivalis (accession number AY130295) not published yet and with a
partial ITS-1 sequence (accession number AJ416956) described as summer
bolete by Moor et al. (2002).
Sequence variation among Amanita muscaria was found to be generally
very low. In particular the ITS1-5.8S-ITS2 sequence our A. muscaria mycelium
differed by only 1 or 2 bp in the ITS-1 and by 2 or 4 bp in the ITS-2 from A.
muscaria sequences available in the database and its phylogenetic position is
shown in Fig. 5.
Fungal Diversity
59
Fig. 1. Growth trend of the isolated strains on mWPM: Bre1 (B. aestivalis), Blu3 (B. luridus),
Edu2 (B. edulis), Amu1 (A. muscaria) and Hra1 (H. radicosum).
Fig. 2. Morphological characteristics of mycelia. a. Hyphal growth of B. luridus mycelium into
the agar. b. Interlacing hyphae of A. muscaria. c. Hyphal swelling of B. aestivalis. d. Clamp of
A. muscaria. e. Clamp of H. radicosum. f. Hyphal swelling of B. edulis closely a septum. Bars
= 5 µm.
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
012345678
Weeks
Diametral growth (cm)
Bre1
Ed u 2
Blu3
Amu1
Hra1
60
Fig. 3. Phylogenetic position of B. aestivalis mycelium (Bre1 strain), B. edulis mycelium
(Edu2 strain), B. luridus mycelium (Blu3 strain). Tree inferred by maximum likelihood
analysis based on rDNA sequences, including the ITS-1, 5.8S and ITS-2 regions (Ln likelihood
= -7166.58725). Pisolithus tinctorius was used as outgroup. The numbers below the branches
indicate the percentage at which a given branch was supported in 200 bootstrap replications.
0.1
B. luridus (AJ419191)
B. luridus fruit body (AY278766)
B. luridus (str. Blu3) mycelium (AY278765)
Uncultured ectomycorrhiza - Boletaceae (AF465183)
B. rhodoxanthus (AJ419189)
B. calopus (AJ296293)
B. fragrans (AJ419186)
B. erythropus (AJ496595)
B. impolitus (AJ419187)
Pisolithus tinctorius (AF374710)
B. aestivalis (str. Bre1) mycelium (AY278769)
B. aestivalis (AY130295)
AJ419190B
AJ419182B
AF074921B
B. edulis (AJ419182)
B. pinicola (AJ419190)
B. edulis (AF074921)
B. edulis (str. Edu2) mycelium (AY278764)
94
96
100
89
86
90
90
78
72
100
94
89
96
Fungal Diversity
61
Fig. 4. Boletus edulis ITS-1 region sequence alignment. Boletus aestivalis mycelium (Bre1 strain) - AY278769; "Chinese" B. edulis -
AJ416955; B. edulis - AF074921; B. edulis - AJ419182.
AY278769 :
AJ416955 :
AF074921 :
AJ419182 :
* 20 * 40 * 60 * 80
AAGGATCATTATCGAGT---TAGACC-GGGAAGG-GG-------------------------------------------
AAGGATCATTATCGAGT---TAGACC-GGGAAGG-GG-------------------------------------------
AAGGATCATTATCGAAT-CCT--ACCAGGGAGGGAGGGAAAATGGACAAGGACTCTCAAGGCTGTCGCCGGCAACGTGCA
------CATTATCGAATCCCT--ACCAGGGAGGGAGGGAAAATGGACAAGGACCCTCAAGGCTGTCGCCGGCAACGTGCA
aaggatCATTATCGA T T ACC GGGA GG GG
: 32
: 32
: 77
: 72
AY278769 :
AJ416955 :
AF074921 :
AJ419182 :
* 100 * 120 * 140 * 160
----------TTTCCTCGGAC-TCTCCTTCCTAGTTTTCCTTATTTCACCTGTGCACCCTCTGTAGGCCCTCGAGAGAGG
----------TTTCCTCGGAC-TCTCCTTCCTAGTTTTCCTTATTTCACCTGTGCACCCTCTGTAGGCCCTCGAGAGAGG
CGCCTTCCTCTTTCAT-GGACC-CCCCTTTCTAGTTT-CCTTATC-CACCTGTGCACCCTTTGTAGGCCCTCGAAAGAGG
CGCCTTCCTCTTTCAT-GGA-C-CCCCTTTCTAGTTT-CCTTATC-CACCTGTGCACCCTTTGTAGGCCCTCGAAAGAGG
TTTC T GGAc C CCTT CTAGTTT CCTTAT CACCTGTGCACCCT TGTAGGCCCTCGA AGAGG
: 101
: 101
: 153
: 147
AY278769 :
AJ416955 :
AF074921 :
AJ419182 :
* 180 * 200 * 220 * 240
ATCTATGTTTTCTATAATCTA-CT-CTATCGCATGT----CCAGAACGTATACATACAAACTTTTACAACTTTCAGCAAC
ATCTATGTTTTCTATAATCTA-CT-CTATCGCATGT----CCAGAACGTATACATACAAACTTTTA--------------
TTCTATGTTTT---TA-TCTATCTACTACCACATGTATGTCCAGAATGTATACA----AA-TTTTACAACTTTCAGCAAC
TTCTATG--TT---TA-TCTATCTACTACCACATGTATGTCCAGAATGTATACA----AA-TTTTACAACTTTCAGCAAC
TCTATGttTT TA TCTA CT CTA C CATGT CCAGAA GTATACA AA TTTTAcaactttcagcaac
: 175
: 161
: 224
: 216
ITS-1 5.8S
18S rDNA ITS-1
62
Fig. 5. Phylogenetic position of A. muscaria mycelium (Amu1 strain). Tree inferred by
maximum likelihood analysis based on rDNA sequences, including the ITS-1, 5.8S and ITS-2
regions (Ln likelihood = -5929.64239). Agrocybe praecox was used as outgroup. The numbers
below the branches indicate the percentage at which a given branch was supported in 200
bootstrap replications.
There was also little sequence variation among Hebeloma spp. In this
study the ITSs from H. radicosum mycelium differed only by 1 bp in the ITS-1
and by 1 bp in the ITS-2 with the ITS sequences belonging to H. radicosum
(AF124700) submitted to the Genbank by Aanen et al. (2000). The
phylogenetic position of the sample of Hebeloma collected in Italy is presented
in Fig. 6.
0.1 Agrocybe praecox (A F124713)
A. muscaria (AF085490)
A. muscaria (AB081295)
A. muscaria (AJ549964)
A. muscaria (str. Amu1) mycelium (AY278768)
A. muscaria (Z54294)
A. gemmata (AF335440 )
A. pantherina (AB080978)
A. pantherina (AB015701)
A. rubescens (AF085484)
A. rubescens (AB015682)
A. spissa (AF085486)
A.citrina (AB015679)
A. citrina (AF085489)
A. citrina (AY325846)
A. phalloides (AY325836)
A. phalloides (AJ308097)
A. virosa (AB015676)
A. virosa (AY325829)
A. caesarea (AY486237)
94
98
100
100
84
82
90
100
89
90
84
100
87
90
92
89
94
Fungal Diversity
63
The topologies of the phylogenetic trees produced by distance matrix and
parsimony criterions based on this data set agreed with that of the maximum
likelihood.
The robustness of the different branches of interest was confirmed by
significant bootstrap values (> 80%) except for the B. edulis and B. aestivalis
cluster (< 75%).
Discussion
Ectomycorrhizal fungi have been widely studied in different parts of the
world and have considerable economic and ecological importance. For this
reason, these species are often used both for experimental and applied purposes
(Hall and Wang, 1998; Ohta, 1998; Yamanaka et al., 2000). This paper uses
both morphological and molecular methods to characterise the mycelia of these
ectomycorrhizal fungi.
Although morphological characters have been shown to be able to
distinguish the culture of different genera of the isolated fungi, it was difficult
to differentiate between the three Boletus species examined in this study on the
basis of the colony morphology and hyphal characters.
As described by Torres and Honrubia (1991) on PDA and on other media,
Amanita muscaria culture has a regular edge, white mycelium and without
exuded pigments. We also found that A. muscaria hyphae did not grow into the
solid media and its mycelium develop mainly in contact with air on the upper
layer of floating inocula on liquid mWPM (data not shown) probably because
this species needs an oxygen rich environment.
Hebeloma radicosum and Amanita muscaria colonies showed evident
clamped septa, a feature rare in Boletus spp. (Del Vesco, 1963; Scurti et al.,
1978-79) and never observed in our Boletus strains. However, these characters
seem to vary according to the fungus life stage and environmental conditions,
since some Authors (Tozzi et al., 1980-81; Brunner et al., 1992) observed a
high clamp frequency in emanating hyphae of mycorrhizae formed by Boletus
species.
Our Boletus mycelia only showed differences in the morphology of the
marginal zone and in the colour of the colonies. All the examined species of
Boletus exuded brown pigments in hsPDA confirming that this is a distinctive
character of Boletus mycelia (Scurti et al., 1978-79).
The analysis of ITS sequences made it possible to confirm the identity of
our mycelium cultures (Figs. 3-6). To date, however, there is a limited number
of ITS rDNA sequences from Boletaceae in public databases, and few of them
cover the entire ITS length. In this study, it was therefore not possible to resolve
64
the exact taxonomic affiliation of our isolate of the B. edulis species complex.
The scarcity of ITS sequences in the database also prevented a taxonomical
attribution of a member of the B. edulis species group growing in New Zealand
(Stringer et al., 2001). Clearly, molecular data from additional samples from
different geographical origin is still needed to resolve the taxonomy of B. edulis
and allied species.
Among the available ITS sequences of the edible bolete, B. edulis
(accession number AJ416955) described by Moor et al. (2002) contains the
ITS-1 region of a sample from China that shows the highest score of similarity
with the ITS sequences we obtained from our mycelium of B. aestivalis (Fig.
4). Moor describes the molecular difference among the ITS-1 region of some
European Boletus edulis with a Chinese Boletus edulis (61 bp of insertion) to
highlight the presence of "Chinese king bolete" on the market and the possible
target for fraudulent labelling. Unfortunately in his paper there is some
misidentification with the GenBank numbering: the ITS sequence AJ416954
presented as B. edulis (Europe) is identical (274/274 nt = 100% similarity) to
the summer bolete (B. aestivalis AJ416956) listed in the same paper. Thus the
original sequence of European B. edulis described by Moor is missing and the
similarity of the Italian B. aestivalis sequence to the Chinese B. edulis leads to
further discussion and deeper investigation into the origin and polymorphism
among the Italian and Chinese king boletes.
ITS typing is a useful tool for identifying both isolated mycelia and
ectomycorrhizae collected in the field. In fact, the comparison of the ITS
sequences obtained in our research with the sequences available in the database
made it possible to affiliate an uncultured ectomycorrhiza of Boletaceae to the
B. luridus species. As suggested by Horton and Bruns (2001), if all researchers
deposited ITS sequences from at least the major species found in their studies,
this would greatly increase the chance of eventually identifying these species
and would also increase the comparability of species lists across studies.
In this paper we have highlighted the need for the reliability of DNA
sequences deposited in public databases, as recently also suggested by several
Authors (Crous, 2002; Deckert et al., 2002).
Our results also indicate that although it is relatively easy to isolate and
culture these ectomycorrhyzal fungi, it is necessary to use molecular techniques
to avoid misidentification of fungal isolates. It has been shown that
misidentification could occur for the ectomycorrhizal fungi growing slowly in
pure culture such as truffles, Amanita and Boletus species (Mello et al., 2001;
Bridge et al., 2003).
Fungal Diversity
65
Fig. 6. Phylogenetic position of H. radicosum mycelium (Hra1 strain). Tree inferred by
maximum likelihood analysis based on rDNA sequences, including the ITS-1, 5.8S and ITS-2
regions (Ln likelihood = -2445.44712). Agrocybe praecox was used as outgroup. The numbers
below the branches indicate the percentage at which a given branch was supported in 200
bootstrap replications.
0.1 Agrocybe praecox
(AF124713)
H. truncatum (AF124701)
H. sinapizans (AF124682)
H. sinapizans (AY320380)
H. radicosum (str. Hra1) mycelium (AY278767)
H. radicosum (AF124700)
H. mesophaeum (AY311521)
H. mesophaeum (AF124692)
H. senescens (= H. edurum) (AY312987)
H. edurum (= H. senescens) (AF124698)
H. sarcophyllum (AF124715)
H. sacchariolens (AY312985)
H. sacchariolens (AF124689)
H. pallidoluctuosum (AY311526)
H. pusillum (AY312982)
H. crustuliniforme (AF124708)
H. pusillum (AF124702)
H. helodes (AY311516)
H. helodes (AF124710)
100
100
82
96
84
100
100
100
100
85
78
84
96
80
66
Acknowledgements
We thank Dr. Ian Hall for helpful suggestions and drafting the English version of the
manuscript. This study was supported by the CNR Strategic Project: “Tuber: Biotecnologia dei
funghi eduli ectomicorrizici".
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(Received 30 April 2004; accepted 20 December 2004)
