Molecular and Cellular Biochemistry 218: 65–70, 2001.

© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

65

Effects of different carbohydrate sources on the

growth of Tuber borchii Vittad. mycelium strains
in pure culture
Paola Ceccaroli,1 Roberta Saltarelli,1 Paola Cesari,1 Alessandra
Zambonelli2 and Vilberto Stocchi1
1
Istituto di Chimica Biologica ‘Giorgio Fornaini’, Università degli Studi di Urbino, Urbino (PU); 2Dipartimento di
Protezione e Valorizzazione Agroalimentare, Università di Bologna, Bologna, Italy

Received 14 July 2000; accepted 16 November 2000

Abstract
The influence of carbohydrate utilisation on the growth of three strains of Tuber borchii Vittad. mycelium (1BO, 17BO and
10RA) in culture was assessed using culture media containing glucose (control), mannose or mannitol. Mannose was the best
substrate for growth of the strains and this was particularly evident for strain 17BO. Mannitol instead was metabolized only by
10RA and 1BO. In order to explain the different growth trends, analyses of enzyme levels, kinetic parameters, protein patterns
and the morphology of the three strains were carried out. Our results show that these strains of T. borchii mycelium were af-
fected by the substrates used in the media. The aim of the present work was to optimise the in vitro production of T. borchii
mycelium for use in experiments which require the fungus in precise and reproducible conditions, such as mycorrhizal synthe-
sis or protein and nucleic acid extractions. (Mol Cell Biochem 218: 65–70, 2001)

Key words: hexokinase, mannitol cycle, mycelium, sugars, Tuber borchii Vittad.

Introduction fructose as carbohydrate sources but grows poorly, if at all,

Tuber borchii Vittad. is an ectomycorrhizal ascomycete which
forms mycorrhizas with a variety of hosts [1]. The slowness
with which the fungus grows in vitro has hampered the study
of its physiology and limited its ability to synthetise my-
corrhizas under controlled conditions. Some studies regard-
ing the use of different nutrient sources and culture media to
optimise the growth of ectomycorrhizal fungi have been re-
ported in the literature and, in particular, mycelium develop-
ment has been assessed in relation to the carbon, nitrogen and
phosphorus supply [2–5]. As regards mycelial carbohydrate
utilisation, it was observed that ectomycorrhizal fungi utilise
above all glucose and fructose [6], although sucrose and man-
nose each allowed substantial growth for T. melanosporum [7].
In a previous study [8] using only T. borchii mycelium strain
1BO (ATCC 96540) we showed that it utilises glucose and
in sucrose. Subsequently [9] we reported stunted growth in
glucose of three other T. borchii mycelium strains (10RA,
17BO and Z43) as compared to strain ATCC 96540. These
four fungal isolates belong to T. borchii [10], although a ge-
netic analysis demonstrated intra-specific variability [9, 11].
The aim of the current paper, which reports the utilisation
of mannose or mannitol as carbohydrate sources in culture
by T. borchii strains, was to identify the best conditions for
the production of high-quality inoculum for all of the strains
studied. In fact, optimal production of T. borchii mycelium
is a fundamental step in the study of fungus metabolism, of
the interaction between the truffle and its environment, of ge-
netic variability and to obtain mycorrhization under control-
led conditions. In this study carbohydrate utilisation was
evaluated, using a protein assay to compare the growth lev-
els obtained. Furthermore, the enzymatic activities involved

Address for offprints: P. Ceccaroli, Istituto di Chimica Biologica ‘Giorgio Fornaini,’ Via A. Saffi, 2, Università degli Studi di Urbino, 61029 Urbino (PU), Italy
66
in sugar metabolism, i.e. hexokinase and the mannitol cycle
were investigated and sodium dodecyl sulphate-polyacryla-
mide electrophoresis (SDS-PAGE) was performed. Finally,
fungus morphology was evaluated using light microscopy to
identify possible modifications related to the substrates used
in the culture media.
Materials and methods
Materials
Coenzymes, enzymes, substrates and dithiothreitol (DTT)
were purchased from Sigma (St. Louis, MO, USA); β-mer-
captoethanol (β-MSH) from Fluka. Potato Dextrose Agar
(PDA) was obtained from Difco (Detroit, MI, USA). The
culture media for mycelium growth were filtered through
a STERIVEX-GS 0.22 µm Millipore filter (Bedford, MA,
USA). Low molecular weight proteins were obtained from
Bio-Rad Laboratories (Hercules, CA, USA).
Growth of mycelium
Four different mycelium strains, designated 1BO (ATCC
96540), 17BO, 10RA and Z43 were isolated from fresh T.
borchii fruitbodies as described in Saltarelli et al. [9]. (Dried
voucher specimens and mycelia are preserved at the ‘Di-
partimento di Protezione e Valorizzazione Agroalimentare’ of
the University of Bologna, Italy). Isolates were grown in
modified Melin-Norkrans nutrient solution (MMN) (pH 6.6)
according to the method of Molina [12]. Mycelia were cul-
tured in 100 ml flasks, each containing 70 ml of medium
inoculated with fungus cultured in PDA plugs, and kept in a
growth chamber at 24°C in the dark with no agitation. The
four mycelial strains were sampled at set times (15, 30, 50
and 70 days) and processed as reported in Saltarelli et al.
[8] in order to evaluate growth. The sugar concentration was
10 g l–1 of medium for all of the carbohydrates tested.
Sample preparation
After 50 days of growth, the mycelia were harvested, washed
with distilled water to remove traces of the growth medium
and homogenised (0.5 g ml–1) using a Potter homogenizer
with a glass pestle (Steroglass, Italy) in 5 mM sodium-po-
tassium phosphate buffer, pH 8.1, containing 3 mM KF, 3 mM
β-MSH and 1 mM DTT. The suspensions obtained were
then centrifuged at 17,500 g for 15 min to remove broken
hyphae. The supernatant was used for all the experiments
described.
Enzyme assay
The levels of hexokinase (EC 2.7.1.1) activity were measured
spectrophotometrically at 37°C in a system coupled with
glucose-6-phosphate dehydrogenase (EC 1.1.1.49) (0.5 U
ml–1) as described by Beutler [13] with the exception of the
glucose concentration, which was increased to 100 mM.
When mannose (final concentration 100 mM) was used as
substrate, phosphomannose isomerase (EC 5.3.1.8) and
phosphoglucose isomerase (EC 5.3.1.9) were added to the
assay mixture (0.5 U ml–1) to convert mannose 6-phosphate
to fructose 6-phosphate and fructose 6-phosphate to glucose
6-phosphate. In the determination of enzymatic parameters
the final substrate concentrations varied from 0.4–2 mM for
glucose and mannose. One unit of hexokinase activity is
defined as the amount of enzyme which catalyzes the forma-
tion of 1 µmol of glucose 6-phosphate/min at 37°C. Manni-
tol dehydrogenase (MDH) (NADP) (EC 1.1.1.138), mannitol
1-phosphate dehydrogenase (M1PDH) (EC 1.1.1.17) and
mannitol 1-phosphatase (M1Pase) (EC 3.1.3.22) were as-
sayed using the methods described in Ramstedt et al. [14].
The protein concentration was determined according to the
method of Bradford [15].
Sodium dodecyl sulphate-polyacrilamide gel
electrophoresis (SDS-PAGE)
The homogenates obtained as described above were re-
suspended in 15 µl of 2% (v/v) SDS and analysed using verti-
cal slab gels (16 cm × 18 cm × 1 mm) as described in Saltarelli
et al. [9].
Morphological examination
The mycelia were grown for 20 days on a cellophane film (PT
35, Safta, Piacenza, Italy) on MMN agar medium containing
glucose, mannose or mannitol. Small square pieces of cello-
phane bearing growing colonies were cut out and fixed in 0.1
M phosphate-buffered 2.5% glutaraldehyde, pH 7.0, for 1 h
at room temperature, washed, stained with blue Cotton dye in
lactic acid, washed and mounted in lactic acid. The samples
were observed with a Leitz light microscope, at 100 ×.
Results
Mycelium growth on different carbohydrate sources
In the present study we used mannose or mannitol in the liq-
uid media in order to obtain an increased fungal biomass for
different T. borchii mycelial strains. As reported in Fig. 1,

strains 1BO and 10RA grow in mannose or mannitol equally
well as in glucose. In fact, statistical analysis performed at
50 days of growth showed that for each individual strain, the
mean values obtained in repeated experiments were not sig-
nificantly different for the three sugars tested (p > 0.05). On
the contrary, mannose was the optimal carbohydrate source
for strain 17BO, whereas mannitol did not give rise to sub-
stantial protein accumulation (p < 0.05). Only strain Z43 grew
poorly in all the substrates tested (not shown). To increase
Fig. 1. Growth of Tuber borchii mycelium strains in MMN medium con-
taining glucose (l–l), mannose (s–s) or mannitol (O–O) as substrate.
Statistically different curves are indicated by an asterisk (p < 0.05; non para-
metric analysis by Kruskal-Wallis one-way ANOVA).
67
the fungal biomass of Z43 we utilised the disaccharide mal-
tose and the polysaccharide potato starch; neither of these
compounds was however utilised by this strain (not shown).
In order to explain the different carbohydrate utilisation ob-
served especially for strain 17BO, we evaluated the affinity
of hexokinase for its substrates (glucose and mannose) and
the enzymes of the mannitol cycle. As shown in Table 1, the
parameter Vmax/Km for glucose and mannose showed similar
values; thus it can not explain the differences in growth of
17BO strain in these two sugars. In order to interpret the poor
growth of strain 17BO cultured in mannitol we assayed the
enzymes of the mannitol cycle (Table 2). These enzymatic
activities were also investigated in strain 1BO, which grew
very well in the medium containing mannitol and which,
using NMR spectrometry, was found to accumulate manni-
tol as carbohydrate stores (Ceccaroli et al., submitted). As
shown in Table 2, strain 1BO contains all the enzymes of the
complete mannitol cycle whereas, like the growth level, the
enzymatic assays of 17BO strain showed that only fruc-
tokinase activity was similar for both strains whereas M1PDH
activity was three times lower in strain 17BO, M1Pase was
not detectable and MDH was present in lower amounts.
Morphological examination
The three T. borchii strains were also grown in MMN agar
containing glucose, mannose or mannitol for use in morpho-
logical examinations. Remarkably, strain 10RA, the growth
level of which did not seem to be strongly affected by the
different carbohydrate sources utilised in the liquid media,
presented the greatest changes in morphology with respect
to the other two strains analysed. When cultured in glucose,
strain 10RA showed a loose mycelium with poorly ramified
hyphae and grew on the surface of the agar (Fig. 2a). When
Table 1. Affinity of hexokinase from different strains of mycelium for glu-
cose and mannose
a b
Km (mM) Vmax (%) Vmax/Km (%)
17BO
D-(+)-Glucose 0.43 ± 0.080 100 100
D-(+)-Mannose 0.27 ± 0.02 88 ± 5 140 ± 18
1BO
D-(+)-Glucose 0.4 ± 0.1 100 100
D-(+)-Mannose 0.31 ± 0.05 87 ± 15 112 ± 29
10RA
D-(+)-Glucose 0.77 ± 0.1 100 100
D-(+)-Mannose 0.7 ± 0.15 97 ± 15 106 ± 28
a
Maximum velocities are expressed vs. the Vmax for glucose (100%), the
sugar generally utilized in the assay determinations. bThe ratio Vmax/Km was
obtained considering the Km values for glucose as 100%. Data shown rep-
resent the means ± S.E. of 3 independent experiments.
68

gave similar electrophoretic profiles in all of the sugars tested.

Table 2. Enzymes of the mannitol cycle in 1BO and 17BO strains Strain 17BO, when grown in mannitol, presented an electro-
a
phoretic profile with weaker bands compared with its pro-
1BO 17BO
file after growth in glucose and mannose. Finally, strain 10RA
Enzyme (U/mg of proteins) grown in mannose presented an electrophoretic profile with
Fructokinase 0.62 ± 0.06 0.74 ± 0.1 a larger number of bands than when it was grown in glucose
b
M1PDH 0.52 ± 0.06 0.19 ± 0.03
MDH 0.22 ± 0.01 0.021 ± 0.005
or mannitol and showed an interesting band of around 31 kDa
M1Pase 0.47 ± 0.028 0

The values reported in this Table are the means ± S.E. of 3 independent
determinations. aTo perform the enzymatic assays mycelia were harvested
from 10 culture flasks; bM1PDH – mannitol 1-phosphate dehydrogenase;
MDH – mannitol dehydrogenase; M1Pase – mannitol 1-phosphatase.

this strain was grown in mannose or mannitol, it was very

branched and showed an increase in apical hyphal branch-
ing. In mannitol, over 75% of the branches were dichotomous
(Fig. 2b); on the contrary, in mannose, only one of the two
daughter hyphae developed after the apical branching and the
mycelium had an irregularly undulated appearance (Fig. 2c).
Strain 17BO grew on the agar surface with all of the sub-
strates except mannitol and forms rich hyphal anastomoses
with an H-shaped structure, a typical feature of mycelial
Tuber species [16]. In all the sugars tested, 1BO showed a
compact texture (Fig. 2, 1BO), with the hyphae less extended
and more branched than those of other strains.

Electrophoretic analysis

Subsequently, the electrophoretic profiles of the three strains

grown in different sugars were analysed (Fig. 3). Strain 1BO

Fig. 2. SDS-PAGE of crude extracts from three T. borchii strains at 50
days of growth in different carbohydrate sources: G – glucose; Ms – mannose;
Ml – mannitol. 2.5 µg of total proteins for each samples were loaded onto
the gel. The single lane (Ms*) represents 17BO strain cultured in mannose
at 30 days of growth. Low molecular weight proteins were used as stand-
ard.
Fig. 3. (a) Strain 10RA grown in glucose showed a loose mycelium with
poorly ramified hyphae; bar: 20 µm. (b) Strain 10RA grown in mannitol
presented dichotomous branches (arrow); bar: 10 µm. (c) Strain 10RA when
grown in mannose, only one of the two daughter hyphae developed after
the apical branching (arrow); bar: 10 µm. (1BO) Strain 1BO grown in glu-
cose showed a compact texture; bar: 20 µm.

which seems to be differentially expressed. This band also
appeared in strains 1BO and 17BO when grown in mannose
as carbohydrate source. Strain 17BO presented slow growth
in mannose for the first 30 days and then utilized more read-
ily only this sugar. At 30 days of growth, the electrophoretic
profile of 17BO cultured in mannose showed a weaker band
than that present in the same profile at 50 days.
Discussion
This work is the first examination of utilisation of mannose
and mannitol as carbohydrate sources by different strains of
T. borchii mycelium in pure culture. This mycorrhizal asco-
mycete usually utilises glucose or fructose as carbohydrate
source, but the slowness with which the fungus grows in vitro
led us to test other carbohydrate sources in an attempt to
optimize its growth level. The results reported in this paper
show that the strains analysed present different trends de-
pending on the carbohydrate source utilised in culture. These
T. borchii strains, and especially strain 17BO, utilised pref-
erentially mannose, in agreement with what was found for
the ectomycorrhizal fungus T. melanosporum [7]. Since
mannose is phosphorylated by hexokinase [17], we tried to
explain the preference for mannose rather than glucose by
assaying the affinity of hexokinase for these two sugars.
Hexokinase is of fundamental importance in the metabolism
of sugars [18] and has been implicated as glucose sensor for
glucose-repressible genes in eukaryotes such as yeast [19],
plants [20] and mammals [21]. In ectomycorrhizal fungi,
glucose 6-phosphate enters both the pentose phosphate and
glycolytic pathways and is incorporated into the mannitol
cycle via fructose 6-phosphate [22, 23]. The Vmax/Km values
for glucose and mannose reported in Table 1 do not justify
completely the different growth levels. Mamoun and Olivier
[7] suggested that the higher growth of T. melanosporum in
mannose may be due to the fact that the fungus had adapted
to its environment, since mannose is an important constitu-
ent of plant glycoproteins. In this work we report that the
electrophoretic profile of T. borchii strains grown in mannose
presented a characteristic band at 31 kDa, which could be
involved in the mannose metabolism of these strains.
As regards mannitol utilization, it was reported in the lit-
erature that this compound was the main soluble carbohydrate
in ascomycetous fungi, for which it serves as a carbon store
and as a form of stored reducing power [24]. The results re-
ported in Table 2 show that strain 1BO, which grows in man-
nitol, expresses all the enzymes of the mannitol cycle. These
data are in agreement with those obtained for other ecto-
mycorrhizal fungi such as S. brunnea and Cenococcum
graniforme [22]. On the contrary, 17BO, which grows poorly
in mannitol, lacks mannitol phosphatase and shows low man-
nitol dehydrogenase activity which is, however, probably
69
sufficient to produce fructose which, via fructokinase, enters
the glycolytic pathway. In fact, when strain 17BO was grown
in mannitol it expressed all the enzymatic activities of the
Embden-Meyrhof pathway (data not shown).
Finally, the morphological data show that the mycelial
strains were influenced by the sugars utilised in the solid
culture. The hyphal morphology of the strain 1BO was little
influenced by the carbohydrate source whereas the strains
17BO and in particular the strain 10RA showed alterations
in branching when they were grown in mannose and manni-
tol. Our data are in agreement which has been reported with
other fungal species that sugars can modify the hyphal mor-
phology also when they apparently do not have any effect on
growth rate [25].
The differences observed among these strains of the same
species, should be considered in order to select the best com-
bination strain-substratum in order to obtain an high-quality
inoculum. This step could be necessary for the future produc-
tion in large-scale of T. borchii mycelium in order to extract
an advantage in the biotechnology applications.
Acknowledgements
This work was supported by the Regione Marche CEE 2081/
93 DOCUP OB. 5B-Misura 1. 1. 3.: Produzioni di qualità,
azione 5, sperimentazione e tartuficoltura.
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