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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.
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Fungal Diversity
Morphological and molecular characterisation of mycelia of
ectomycorrhizal fungi in pure culture
Mirco Iotti1, Elena Barbieri2, Vilberto Stocchi2 and Alessandra
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-
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.
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:
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
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.
Collection locality Probable host plant Date of
Boletus aestivalis
Boletus edulis
Boletus luridus
Boletus luridus
Amanita muscaria
Hebeloma radicosum
Castiglione dei Pepoli
Castiglione dei Pepoli
Castiglione dei Pepoli
Castanea sativa Mill.
Fagus silvatica L.
Castanea sativa Mill.
Castanea sativa Mill.
Fagus silvatica L.
Fagus silvatica L.
Table 2. Composition of mWPM.
Compound Concentration Compound Concentration
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
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
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 ( 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
(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).
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.
Table 3. Species analysed in this study.
Species GenBank
accession no.
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.
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 &
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
Table 3 continued. Species analysed in this study.
Species GenBank
accession no.
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 &
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
Amu1 appressed appressed even even felty felty white white slightly
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
diameter (µm)
427.90 ± 159
473.14 ± 168.12
466.91 ± 165.01
462.20 ± 206.12
426.75 ± 256.11
54.76 ± 14.35
32.40 ± 10.57
40.72 ± 10.72
59.52 ± 13.46
58.67 ± 17.09
78.82 ± 20.94
82.71 ± 27.84
85.25 ± 25.30
40.15 ± 14.07
46.19 ± 13.38
2.47 ± 0.23
2.75 ± 0.27
2.35 ± 0.24
1.99 ± 0.21
2.22 ± 0.25
Data are the mean of 50 measures from three different Petri dishes.
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
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.
Diametral growth (cm)
Ed u 2
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.
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)
B. edulis (AJ419182)
B. pinicola (AJ419190)
B. edulis (AF074921)
B. edulis (str. Edu2) mycelium (AY278764)
Fungal Diversity
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
: 32
: 32
: 77
: 72
AY278769 :
AJ416955 :
AF074921 :
AJ419182 :
* 100 * 120 * 140 * 160
: 101
: 101
: 153
: 147
AY278769 :
AJ416955 :
AF074921 :
AJ419182 :
* 180 * 200 * 220 * 240
: 175
: 161
: 224
: 216
ITS-1 5.8S
18S rDNA ITS-1
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)
Fungal Diversity
The topologies of the phylogenetic trees produced by distance matrix and
parsimony criterions based on this data set agreed with that of the maximum
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%).
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
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
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
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
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)
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)
... General technical works for the identification of ectomycorrhizal fungi are those of Singer (1986), Arora 1986, Moser (1978, Ainsworth et al. (1973a, b), Watling and Gregory (1980), Kumar et al. (1990), Lakhanpal (1996), Natarajan and Raaman (1983), Lakhanpal et al. (2010) and Das and Sharma 2005. Over the past few years there has been a dramatic increase in the number of studies on ectomycorrhizal (ECM) fungi involving molecular identification of species and individuals (Horton and Bruns 2001;Horton 2002;Iotti et al. 2005;Toftegaard et al. 2010). Janowski et al. (2019) pointed out that morphological identification of the ectomycorrhizae often proves to be misleading and now a number of molecular methods that require isolation of nucleic acids are being used. ...
This chapter attempts to trace the developments in ectomycorrhizal (ECM) research in India. The research on ECM in India was initiated at erstwhile FRI (now ICFRE) at Dehradun after independence in the late fifties by Dr. B.K. Bakshi and collaborators. They described the ECM in some important forest trees, viz., Pines, Deodar and high-altitude conifers. They isolated the mycorrhizal associates in pure culture and artificially inoculated the seedlings in a few cases. More impetus to ECM research was provided in the late seventies and early eighties from South, North and Eastern India. Natarajan, Raaman, Reddy and Mohan and their associates intensely investigated the ECM and its implications in seedling regeneration in Pinus patula and other pines. Sharma, Mishra and their students carried out similar studies on P. kesiya in the eastern Himalayas. Lakhanpal and collaborators studied the various aspects of ECM in Chir Pine, Blue Pine, Deodar, Fir, Spruce, Chilgoza Pine, Yew and apple plants in N.W. Himalaya. All these studies concerned characterization, identification, mycobiont association, physical and chemical status and artificial inoculation with selected mycobionts. Recently (2019, 2020) Atri and his students have carried out similar studies on ECM of sal trees in H.P. Tapwal and associates at HFRI, Shimla, are working on the ECM relationship of some important Himalayan forest tree species. The emphasis has gradually shifted now towards evaluation of field performance of the inoculated seedlings which is the primary aim of studies on this symbiotic association.
... It is an ectomycorrhizal fungus and it is usually associated with Angiospermae trees, such as Betula, Castanea, Cistus, and Quercus species, and also coniferous ones, such as Abies, Picea, and Pinus [5,8]. For this reason, on this day, it is impossible to grow A. muscaria basidiocarps in a laboratory, although it is possible to grow mycelium on synthetic media, despite a very slow growth rate [9]. A comparative genome analysis of the mitochondrial DNA suggested a common origin of the ectomycorrhizal Amanita, forming a distinct clade from the saprophyte and, therefore, non-symbiotic Amanita [10]. ...
Full-text available
Amanita muscaria is the most emblematic mushroom in the popular representation. It is an ectomycorrhizal fungus endemic to the cold ecosystems of the northern hemisphere. The basidiocarp contains isoxazoles compounds that have specific actions on the central nervous system, including hallucinations. For this reason, it is considered an important entheogenic mushroom in different cultures whose remnants are still visible in some modern-day European traditions. In Siberian civilizations, it has been consumed for religious and recreational purposes for millennia, as it was the only inebriant in this region.
... To find phylogenetic relationship in mushrooms, ITS sequences of these mushrooms are compared with those sequences already deposited in the GenBank database. Published sequences can also be retrived from different databases and compared with the ITS sequences of fungi whose phylogenetic relationship have to be find (Iotti et al., 2005;Neves et al., 2012). Identification of any mushroom depends on the similarities between known and unknown sequences (Frøslev et al., 2007;Para, 2008). ...
Taxonomy and phylogenetics study of mushrooms from District Bagh, Azad Jammu and Kashmir, Pakistan
... This pattern is also supported by sequence data published for these species (see references in Table 1 and Iotti et al. 2005;Slot et al. 2010;Kasuya et al. 2013Kasuya et al. , 2017Maeno et al. 2016;Vu et al. 2019; as well as data published by NRBC (National Biological Resources Collection) of NITE (National Institute of Technology and Evaluation), Japan, accessed via and sequence data of Estonian collections deposited in UNITE, Nilsson et al. 2019). There is one noteworthy occurrence of H. radicosum in Asia, evidenced by a sequence published from a culture isolated in China (45° 21' N, 127° 32' E;Fan & Fuqiang 2011) that clusters with the European H. radicosum sequences. ...
Hebeloma radicosum, known for its long ‘root’ and membranous ring, has regularly been recorded in Japan and been the subject of many studies revolving around ectomycorrhizal fungi exposed to high levels of nitrogen compounds and tripartite associations between fungi, host trees and mammals, including moles, wood mice and shrews. However, the species recorded in Japan is in fact not H. radicosum but a closely related species, also in Hebeloma section Myxocybe, described here as Hebeloma sagarae. This mushroom is macroscopically very similar to Hebeloma radicosum, also with a long ‘root’ and membranous ring, but distinct molecularly and in its distribution. We also examine other ‘rooting’ Hebeloma species from Japan: H. luchuense and H. radicosoides; both are shown to be members of H. sect. Scabrispora. A fourth ‘rooting’ Hebeloma from Japan is shown to be the same as or a close relative of Hebeloma danicum originally described from Europe.
... Several multi-locus molecular phylogenetic studies carried out across the northern hemisphere in the last 20 years, initially at regional level and later at a global scale, have revealed a number of new species-level lineages within Boletus s. str., and many other previously described taxa have been confirmed with robust statistical supports (Simonini et al. 2001;Moor et al. 2002;Iotti et al. 2005;Leonardi et al. 2005;Mello et al. 2006;Zhao et al. 2006;Vizzini et al. 2007;Beugelsdijk et al. 2008;Lian et al. 2008;Korhonen et al. 2009;Dentinger et al. 2010;Yan et al. 2010;Feng et al. 2012;Arora and Frank 2014b;Dentinger and Suz 2014;Fedosova and Kovalenko 2014;Zeng et al. 2014;Cui et al. 2016;Chakraborty et al. 2017;Sarwar et al. 2018;, suggesting that this group is particularly diverse and ecologically important for forest ecosystems. Furthermore, the high number of recently detected phylogenetic species still lacking a scientific name prove that there are likely additional undescribed taxa yet to be discovered and documented, especially from temperate eastern and tropical southeastern Asia and Australasia Wu et al. 2014;Cui et al. 2016). ...
A detailed and comprehensive list of the edible porcini mushrooms occurring in the world is provided. Each species is accompanied by a list of synonyms, pertinent geographic range, data on ecology and symbiotic partnerships, relevant bibliography, and, whenever necessary, by additional taxonomic and/or nomenclatural notes. Phylogenetic species that have been recognized by using molecular analysis but still lack scientific names are also included.
... For their genetic characterization, the cultures were transferred on fresh modified woody plant medium (mWPM) (Iotti et al., 2005) without agar addition and incubated in the dark at 23 AE 1 C for 60 days. ...
Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study we analyzed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behavior of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia. This article is protected by copyright. All rights reserved.
In recent years, there has been a lot of buzz about the possibilities of marine microflora as a source of new therapeutic drugs. The strong anti-tumor potency of compounds found in marine resources reflects the ocean's enormous potential as a source of anticancer therapeutics. In this present investigation, an ambuic acid derivative anticancer compound was isolated from Talaromyces flavus, and its cytotoxicity and apoptosis induction potential were analyzed. T. flavus was identified through morphological and molecular analysis. The various organic solvent extracts of T. flavus grown on different growth mediums were evaluated for cytotoxicity on different cancer cell lines. The potent cytotoxicity was shown in the ethyl acetate extract of a fungal culture grown in the M1-D medium for 21 days. Furthermore, the anticancer compound was identified using preparative thin layer chromatography, followed by its purification in significant proportions using column chromatography. The spectroscopic and chromatographic analysis revealed that the structure of the purified molecules was an ambuic acid derivative. The ambuic acid derivative compound showed potent cytotoxicity on MDA-MB-231 (breast cancer cells) with an IC50 value of 26 μM and induced apoptosis in the MDA-MB-231 cells in a time-dependent and reactive oxygen species-independent manner.
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The family Boletaceae primarily represents ectomycorrhizal fungi, which play an essential ecological role in forest ecosystems. Although the Boletaceae family has been subject to a relatively global and comprehensive history of work, novel species and genera are continually described. During this investigation in northern China, many specimens of boletoid fungi were collected. Based on the study of their morphology and phylogeny, four new species, Butyriboletus pseudoroseoflavus, Butyriboletus subregius, Tengioboletus subglutinosus, and Suillellus lacrymibasidiatus, are introduced. Morphological evidence and phylogenetic analyses of the single or combined dataset (ITS or 28S, rpb1, rpb2, and tef1) confirmed these to be four new species. The evidence and analyses indicated the new species’ relationships with other species within their genera. Detailed descriptions, color photographs, and line drawings are provided. The species of Butyriboletus in China were compared in detail and the worldwide keys of Tengioboletus and Suillellus were given.
Conference Paper
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The first important step in truffle cultivation is the production of Tuber infected plants. For this purpose, three forms of inoculum can be used: spores, infected roots or pure cultures. It is the last of these which shows the most promise because it provides the opportunity for selecting fungal strains with ideal infections, affinity for the host plant and adaption to the ecological conditions, thereby optimising truffle production. Our initial experimental results confirm that strains can vary in infectivity and host specificity. However, before this information can be applied commercially it will be necessary to standardise the inoculation technique, perfect the preparation of the inoculum and select the most suitable medium for growing the cultures. In the future, it should also be possible to select beneficial fungi and bacteria that can promote ectomycorrhizal development and fruit body formation.
Pezizales are a widespread group of fungi, basal to the other filamentous ascomycetes. Most species live in soil as saprobes, in a mycorrhizal relationship with a wide range of plants, or as plant parasites. The lineage Morchellaceae–Discinaceae–Helvellaceae–Tuberaceae includes most of the commercially valuable species in the order. The truffles in the genus Tuber and morels in the genus Morchella arguably command more interest in culinary circles than any other groups of mushrooms. In recent years, the interactions of these fungi with plants have been thoroughly researched although many aspects still need to be clarified. In this chapter, we describe and compare these two groups of mushrooms and take a look at the evidence as to whether there are real trophic differences from those traditionally held and if things are not quite as simple as our forebears would have had us believe. We explore the range of host plants involved in the interactions, the morpho-anatomy of symbiotic structures, the molecular mechanisms of symbiosis, and the influence of other microbial species.
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Phylogenetic relationships within the genus Hebeloma (Cortinariaceae, Agaricales) were determined, based on nuclear ribosomal ITS sequences, using cladistic methods. Special emphasis was on phylogenetic relationships within the H. crustuliniforme complex. In total 52 sequences were analysed, representing 51 collections and 39 taxa. Agrocybe praecox and two species of Alnicola were used as outgroups. The genus Hebeloma appears to be monophyletic. Several well supported clades could be recognized. However, many of the basal relationships are unresolved or only weakly supported. Alternative topologies could not be rejected. It is therefore impossible to derive a revised infrageneric classification of Hebeloma. The H. crustuliniforme complex appears paraphyletic, consisting of two clades with three and 17 intercompatibility groups respectively. In the second clads many of the phylogenetic relationships are also unresolved, reflecting a high rate of recent speciation events. Most of the species in this clade form ectomycorrhizae with members of the Salicaceae. The taxon that is basal to this clade, however, is not associated with these hosts. The host tree switch to Salicaceae has been followed by extensive and rapid speciation.
Interest in cultivating edible ectomycorrhizal mushrooms has grown in the past few decades with the realization that there had been dramatic falls in the availability of some species and that increased demand far exceeded supply. Ectomycorrhizas are formed by plants in a number of significant families such as the Betulaceae, Cistaceae, Dipterocarpaceae, Fagaceae, Myrtaceae, Pinaceae and Salicaceae (e.g. Trappe 1962; Becker 1983; Harley and Smith 1983). As a consequence, such fungi are widely distributed throughout the world. Even in New Zealand, where only six native species form ectomycorrhizal associations (Nothofagus spp. and Leptospermum scoparium J.R. et G. Forst.), extensive plantings of eucalypts, pines, oaks, and the like have ensured that here too ectomycorrhizal fungi are now widespread. Many of these fungi, such as Amanita phalloides (Vaili, ex. Fr.) Secr., produce extremely poisonous fruiting bodies, but others are not only edible but highly sought after by chefs and gourmets and have wellestablished international markets.
Four mycelial strains of Tuber borchii Vittad. were studied in order to find DNA polymorphisms. Polymerase chain reaction-based techniques for assaying differences in the genome were used. Although a very low level of genetic variability was observed, the few polymorphisms found were sufficient to elaborate strain-specific markers suitable for use in biotechnological applications.
To determine the phylogenetic relatedness of Amanita species, internal transcribed spacers (ITSs) and the 5.8S ribosomal RNA gene were amplified by polymerase chain reaction and then sequenced according to the dideoxy chain termination method using an automatic DNA sequencer. The ITS region provided sufficient variability for phylogenetic analyses within the species. Analyses of the ITS sequence data by distance and parsimony methods revealed that the Amanita species are composed of three distinct groups whose main branch is strongly supported by bootstrap analysis. The Singerian system did not fully correspond to present phylogenetic results based on molecular data. The amyloid nature of the spores was still phylogenetically significant and the type of volva as well as the cap color were proved to be additional important characters in Amanita phylogenetics.
The ectomycorrhyzal fungus Boletus reticulatus formed young fruit bodies in pure culture on liquid and solid media. Primordia formation started 31-32 d after inoculation on liquid medium. The primordia developed into the Young fruit bodies with convex pileus and clavate stipe 44 d after inoculation on liquid medium. The ability of this fungus to form fruit bodies declined at one and half years after isolation. Sufficient nutrient in medium is required for the fungus to form mature fruit bodies in pure culture.
Molecular data confirm the exclusion of Rhizopogon melanogastroides from the genus Rhizopogon. These data, as well as the ectomycorrhiza features do not confirm the inclusion of this taxon in the genus Melanogaster.