MINIREVIEW

Tru¥es: much more than a prized and local fungal delicacy

Antonietta Mello1, Claude Murat2 & Paola Bonfante1,2
1
Istituto per la Protezione delle Piante del CNR, Sezione di Torino, Torino, Italy; and 2Dipartimento di Biologia Vegetale dell’Università di Torino,
Torino, Italy

Correspondence: Antonietta Mello, Istituto Abstract

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per la Protezione delle Piante del CNR,
Sezione di Torino, Viale Mattioli, 25, 10125
Torino, Italy. Tel.: 1390116502927; fax:
1390116705962; e-mail: a.mello@ipp.cnr.it
Received 22 November 2005; revised 22 March
2006; accepted 23 March 2006.
First published online 19 April 2006.
doi:10.1111/j.1574-6968.2006.00252.x
Editor: Richard Staples
Keywords
molecular identification; truffle-ground;
environmental microbiology; truffles.
Truffles are hypogeous fungi which live in symbiosis with plant host roots in order
to accomplish their life cycle. Some species, such as Tuber magnatum Pico, the
‘white truffle’, and Tuber melanosporum Vittad., the ‘black truffle’, are highly
appreciated in many countries because of their special taste and smell. The great
demand for the black and white truffles, the increasing attention towards other
species of local interest for the rural economy (such as T. aestivum) together with a
drop in productivity, have stimulated researchers to develop projects for a better
understanding of the ecology of truffles by exploiting the new approaches of
environmental microbiology and molecular ecology. Specific primers have been
developed to identify many morphologically similar species, the distribution of
T. magnatum has been followed in a selected truffle-ground, the phylogeography of
T. melanosporum and T. magnatum has been traced, and the microorganisms
associated with the truffles and their habitats have been identified.

truffles were included in the order Tuberales, together with

Introduction
Truffles were prized by ancient Greeks and Romans as an
Epicurean delight as far back as the Apicius’s legendary
banquet in 20 AD, but the scanning of scientific literature on
truffles also demonstrates a longstanding love affair between
science and the aromatic fungus. The first paper devoted to
the nature of truffles appeared in 1564 (Ciccarelli, 1564),
while other publications have made them a cult food since
1826 (Brillat-Savarin, 1826). Later the scented truffles were
identified as ‘diamonds’ and ‘the best food’ by Hervé This,
who was the inventor of molecular gastronomy. What we
call a truffle is a hypogeous edible fungus that undergoes a
complex life cycle during which the mycelium establishes a
symbiotic interaction (ectomycorrhizae, Fig. 1a) with the
roots of trees, such as oak, poplar, willow and hazel (Harley
& Smith, 1983), and some shrubs, such as Cistus (Fontana &
Giovannetti, 1978–79). As a final step, hyphae aggregate and
develop a fruiting body – the truffle – which is an ascoma
bearing asci and the products of meiotic events, the ascos-
pores (Fig. 1b).
The true truffles belong to the genus Tuber, one of the few
ectomycorrhizal Ascomycetes, even if other Ascomycetes are
considered truffles, such as the desert truffles (Kirk et al.,
2001). While in traditional classification systems the true
all hypogeous ascomycetes, today they are placed in the
order Pezizales. This includes both hypogeous and epigeous
fungi, with either saprotrophic or symbiotic lifestyles, but
which are all related phylogenetically (O’Donnell et al.,
1997; Percudani et al., 1999).
Some species, such as Tuber magnatum Pico, the ‘white
truffle’, and Tuber melanosporum Vittad., the ‘black truffle’,
are in great demand by the food market in many countries
because of their special taste and smell, resulting from a
blend of hundreds of volatile compounds (Bellesia et al.,
1998; Gioacchini et al., 2005). Several biotic (fungi, yeasts,
bacteria, mesofauna, plant host) and abiotic (soil composi-
tion, weather such as rain, sunshine and temperature)
factors could influence truffle life and enhance or inhibit
ascocarp formation (Ceruti et al., 2003). Truffle species
present common ecological features such as a relatively wide
range of host species and the need for a calcareous soil (pH
between 7 and 8), except Tuber borchii tolerating slightly
acidic soils. On the contrary, there are important differences
in their geographic distribution. While T. borchii and
Tuber maculatum are found throughout Europe (Riousset
et al., 2001), T. melanosporum is collected in the South and
West Europe – Italy, France and Spain – and T. magnatum
fruiting bodies have so far been collected in Italy and in the

FEMS Microbiol Lett 260 (2006) 1–8

c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
2 A. Mello et al.
East Europe – Croatia, Slovenia and Hungary – resulting in a
limited availability.
At 300–400 Euros per 100 g during the 2004 fruiting
season, it is clear why T. magnatum fruiting bodies are one
of the most expensive delicacies, together with caviar. From
September to January, truffle hunters search for the piquant
truffles helped by pigs or trained dogs. The great demand for
black and white truffles, the increasing attention towards
other species of local interest for the rural economy (e.g.,
Tuber aestivum) together with their drop in productivity,
stimulated researchers to develop projects for a better
understanding of the ecology of truffles by exploiting the
new approaches of environmental microbiology and mole-
cular ecology.
The aim of this review is to briefly illustrate how many of
the open questions raised by truffle biology regarding
systematics, diagnosis, population genetics and phylogeo-
graphy, and morphogenesis, have been addressed recently by
researchers, taking advantage of the most advanced techni-
ques of molecular biology.
Morphological vs. molecular identification
Truffles live all over the world, distributed especially in many
regions of the northern hemisphere. Around 200 European
species, varieties and forms of Tuber have been described by
mycologists over the centuries (from the 18th to the 20th).
The various events linked to the species and their synonyms
are summarized in a monograph of the European species of
Tuber where the authors consider only 28 species to be valid
(Ceruti et al., 2003).
Truffle fruiting bodies are usually identified from the size
and shape of their spores and asci, spore wall ornamenta-
tion, structure of the peridium and gleba. These features are
generally recognized by specialists; however, sometimes,
identification is unreliable. This is the case for species with
very similar morphological features, such as T. brumale–
T. brumale var. moschatum and T. melanosporum–T. hiemal-
bum (Riousset et al., 2001).
Biochemical tools, such as one-dimensional gel electro-
phoresis with total protein (Mouches et al., 1981; Dupré
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Fig. 1. Tuber magnatum mycorrhizae (a) and
ascospores (b).
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et al., 1985) and isoenzyme analysis (Pacioni & Pomponi,
1991; Gandeboeuf et al., 1994; Urbanelli et al., 1998), were
the first to be used to verify the morphological identification
of truffles. However, these techniques present several pro-
blems: isoenzyme analysis needs high quality material while
total protein analysis depends on physiological status and
cannot be used with mycorrhizae.
In 1993, our laboratory applied, for the first time,
molecular tools for identifying truffles. Multiloci analyses
such as RAPD (random amplified polymorphism DNA) and
RAMS (random amplified microsatellites sequences), re-
spectively, identified six (Lanfranco et al., 1993) and 11
Tuber species (Longato & Bonfante, 1997).
As multiloci techniques also amplified DNA from plant
tissue present in mycorrhizae and from bacteria present in
fruiting bodies, single locus analyses predominate, today, for
Tuber identification.
Specific primers, designed on the internal transcribed
spacer regions (ITS) of ribosomal genes, have been devel-
oped to discriminate T. magnatum from the so-called
‘whitish’ T. borchii and T. maculatum characterized by less
taste and a lower commercial value (Amicucci et al., 1998;
Mello et al., 2000) (Fig. 2). As well as for white truffles,
specific primers distinguish T. melanosporum from T. bru-
male and the Asiatic Tuber indicum, both of which are used
in food in place of T. melanosporum (Rubini et al., 1998;
Douet et al., 2004). Processed foods sold as containing
T. melanosporum can be contaminated with up to 5% of
other Tuber species (Mabru et al., 2004).
Besides ascocarp identification, the identification of the
fungus during its symbiotic phase has been one of the major
topics in truffle research. The molecular methods developed
for fruiting body identification have also provided diagnos-
tic tools to confirm the occurrence of the desired fungus in
the mycorrhizal roots (Stocchi, 1999; Mello et al., 2001;
Rubini et al., 2001) where morphological identification is
more difficult. This is fundamental in order to follow the
fate of inoculated plants.
Although research on the above species was important
because of the high commercial profits involved, another
challenge has been the controversial taxonomic position of
FEMS Microbiol Lett 260 (2006) 1–8
Truffles: much more than a prized and local fungal delicacy 3

st

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eru

tivu
av a
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Fig. 2. Internal transcribed spacer regions am-
8

him

pub
bru
bor

ex c

aes
C1

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ruf
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plification with specific primers P7/M3 from fruit-

pU

T.
T.

T.

T.
T.

T.

T.

T.
T.

T.

T.

T.

T.
T.
T.

T.
body and mycorrhizae of Tuber magnatum and
from different Tuber spp. Only T. magnatum
shows a 434 bp band. First lane: molecular size
434 bp
marker pUC18 DNA HaeIII digest. (Modified from
Fig. 2 in Mello et al., 1999.)

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T. aestivum Vittad. with respect to Tuber uncinatum Chatin.
These taxa also have a moderate commercial value, and
unlike T. magnatum and T. melanosporum, they have a wide
geographic distribution. They have been found as far north
as Gotland Island, in Sweden. The length of their spore
reticulum was considered the most useful morphological
characteristic for distinguishing the two taxa. However,
some ecological features, geographical distributions and
smell and taste are distinctive of the two taxa. These taxa
have been separated by total protein analysis (Mouches
et al., 1981). After two publications leading to controversial
results (Mello et al., 2002; Paolocci et al., 2004), but
providing the first ITS sequences of these taxa, a very recent
work based on a higher number of samples seems to close
the debate: the height of the spore reticulum is not diag-
nostic, and it is not possible to separate T. aestivum from
T. uncinatum (Weden et al., 2005).
The Chinese black truffles (Tuber pseudohimalayense,
Tuber sinense and T. indicum) are other cases where species
limits are still discussed, as illustrated by Zhang et al. (2005).
As for prokaryotes, where assignment of isolates to species is
based on phenotypic measures and genome similarity
(Gevers et al., 2005), similar difficulties exist for defining
the species of fungi (Taylor et al., 1999).
Even if not always possible, the availability of molecular
probes has helped to resolve the problem of interspecific
differences of truffles, limiting frauds, and answering ques-
tions related to their taxonomy but, above all, allowed
researchers to investigate the fungus during its symbiotic
interaction with the trees in the truffle-ground.
From genetic variability to a hint in the
past
Truffles show variation in several traits, including their
organoleptic properties, across their geographical range.
These variations could be due to environmental and/or
genetic factors. In a highly quoted paper, Bertault et al.
(1998) claimed: ‘morphological and organoleptic differences
seen over the geographical range of the black truffles can
probably be explained by environmental variation rather
than by genetic factors.’ However, Murat et al. (2004) were
successful in revealing a strong geographic pattern for T.
melanosporum (FST = 0.20) using the moderate variations of
ITS region of nuclear rRNA gene sequences. A significant
association between genetic (FST) and geographical dis-
tances (Mantel test) was also found when the distance values
between populations in western France and the populations
of the Rhone Valley were clustered in two groups. On the
basis of this significant genetic differentiation and in con-
trast with previous data (Bertault et al., 1998, 2001), it is
suggested that genetic determinants play a role in the
organoleptic differences of the black truffle.
Bertault et al. (1998) explained the low level of poly-
morphism found for T. melanosporum suggesting that it
went through a population bottleneck after which new
allelic haplotypes have originated in low frequencies. During
the maximum expansion of the glaciers, the deciduous
forest of Europe was restricted to the Mediterranean coastal
zone (Bennet et al., 1991). Climatic and fossil data support
the hypothesis that three regions hold the main glacial
refuges for the host trees of T. melanosporum: the Iberian
and Italian Peninsulas, and the Balkans. Since T. melanos-
porum is an ectomycorrhizal symbiont of oaks and other
temperate deciduous trees, such as Tilia and Corylus species,
this symbiotic fungus was likely to have been restricted to
some of these regions. Although pedoclimatic factors likely
influenced T. melanosporum recolonization patterns, two
routes of expansion were proposed for the Perigord truffle in
France closely resembling those of Quercus pubescens (Petit
et al., 2002): the Rhone valley route and the Atlantic route
(Murat et al., 2004, Fig. 3).
Like T. melanosporum, T. magnatum has been the subject
of studies focused on genetic variability (Lanfranco et al.,
1993; Gandeboeuf et al., 1997; Frizzi et al., 2001). Thanks to
the finding of a single nucleotide polymorphism in T.
magnatum ascocarps, it was possible to identify two haplo-
types in a northern Italian population and to trace their
spatial and temporal distribution (Mello et al., 2005, Fig. 4).
Moreover, analysis of samples coming from Italian and
Balkan populations suggested a genetic differentiation in
T. magnatum over its habitat (Mello et al., 2005), a finding

FEMS Microbiol Lett 260 (2006) 1–8

c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
4 A. Mello et al.

More frequent
haplotypes

Oak haplotypes I
Oak haplotype H7
H10-12
II

III

Lorraine
(I,II) Northeastern
regions
Burgundy
(I,II,III)
Jura
(I,VIII)

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Isère
Jura & Pre-Alps
Touraine (I,II)
(I) Drôme
Massif Alps (I,II,IX)
Charentes
(I,II) Central
Western
regions
Perigord- Piedmont,
Quercy (I,II) Italy (I,II,III)

Lower - Rhône-
Provence (I,II) Coastline
Quercy regions of
(I,II) Inner-Provence Southern
Castres (I,II,III) France
(I,II,IV, VII) Languedoc
Pyrenees (II,III,X)
Ariège Oak haplotype
Iberian Rousillon (I)
(I,V,VI) H1
Mountains,
Spain (I,II)
Pyrenees

Fig. 3. Distribution of the 10 internal transcribed spacer (ITS) haplotypes of Tuber melanosporum in France. The pie chart diameters are proportional to
the number of ascocarps analyzed per region. Black lines delimit areas of distribution of the chloroplastic DNA (cpDNA) haplotypes of oaks in France
(Petit et al., 2002): haplotype 1 was found in the southern corner of France; haplotype-7 in the Rhone valley, and haplotypes 10–12 in western France.
Arrowed lines show potential postglacial recolonization routes for the Perigord truffle: the Atlantic red route and the Rhone valley blue route (adapted
from Fig. 2 in Murat et al., 2004).

Sequence characterised amplified region

Fig. 4. Three haplotypes of Tuber magnatum

Haplotype I originated by two single nucleotide polymorph-
isms, indicated by a star in an oligonucleotide
sequence (the different colours indicate nucleo-
Haplotype II tides: T, red; C, blue; G, black; A, green). Two
haplotypes are present in the northern Italian
Haplotype III population (adapted from Fig. 2 in Mello et al.,
2005).

which was further confirmed by Rubini et al. (2005) after an rest of the populations and probably spread from a refuge of
extensive analysis. By polymorphic microsatellites, these T. magnatum in central Italy during the postglacial expansion.
authors found that the southernmost and the northwestern- Unlike the precious black and white truffles, other
most populations were significantly differentiated from the truffle species show a higher genetic diversity, i.e. T. borchii,

c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 260 (2006) 1–8
Published by Blackwell Publishing Ltd. All rights reserved
Truffles: much more than a prized and local fungal delicacy
T. aestivum and T. maculatum (Lanfranco et al., 1993;
Gandeboeuf et al., 1997; Mello et al., 2002; Paolocci et al.,
2004). It is interesting to note that these species have a larger
geographical distribution than T. magnatum and T. mela-
nosporum (Riousset et al., 2001); for example, T. maculatum
is found from France to Russia. It is probable that quatern-
ary climatic modifications had minor effects on them and
consequently the bottleneck has been less important for
them. However, genetic diversity analyses of these species in
their whole geographic distribution are not yet available
leaving many question marks over these hypotheses. An
additional problem arises from the ambiguity of ascocarp
identification in some species: for example the high level of
genetic intraspecific variability so far described in T. borchii
may be related to uncorrected identifications (A. Zambo-
nelli and A. Mello, pers. commun.).
Truffle productivity and truffle-ground
environment
Improving agricultural productivity, through ecosystem
management techniques, has been the first goal of humans
as they had to face the increasing demand for food. Already
in the 16th century, Bruyerin, François I’s physician, re-
ported that the cultivation of the black truffle was possible.
But, truffle cultivation really began in the 18th century,
when Talon planted acorns and harvested truffles near
young trees. The production of truffles increased to 1588
tonnes in France at the end of the 19th century (Chatin,
1869). However, after the first world war, production had
decreased to reach the present-day value of less than 100
tonnes (Callot et al., 1999).
Research programmes for large-scale mycorrhizal pro-
duction have been elaborated in southern European coun-
tries to increase truffle production (Chevalier, 1994). Their
main steps are: production of mycorrhizal roots in con-
trolled conditions, planting out the mycorrhizal seedlings,
checking for the presence of introduced Tuber species
among the ectomycorrhizal symbionts, and harvest of
fruitbodies. Since the 1970s, it has been possible to obtain
mycorrhizal seedlings; they produce T. melanosporum after
5–10 years (Le Tacon et al., 1988). At present, more than
80% of the French production of this truffle comes from
artificial truffle-grounds (http://www.inra.fr/presse/la_
truffe_de_plus_en_plus_rare_et_chere_gare_aux_fraudes).
On the other hand, utilization of inoculated seedlings has
allowed production of truffles in countries where T. mela-
nosporum is not naturally found, such as Israel, the USA and
New Zealand (Ceruti et al., 2003; Hall et al., 2003). At
present, T. melanosporum, T. borchii and T. aestivum are
collected from artificial truffle-grounds. By contrast, pro-
duction of T. magnatum in controlled conditions has been
rarely reported (and it is not certain whether it depends on
FEMS Microbiol Lett 260 (2006) 1–8
5
successful inoculation in the nursery or on infections
established after planting).
Many attempts have been made to isolate truffle mycelia
to produce adequate amounts of inocula, but without
success because of their slow growth. As it is very difficult
to obtain pure mycelial cultures of Tuber spp., truffle-
infected plants are usually produced with spore inoculum.
As a consequence, especially plants inoculated with T.
magnatum spores sometimes became contaminated with
unexpected Tuber spp. or other ectomycorrhizal fungi
(Amicucci et al., 2001). However, only T. borchii mycelium
has been produced in sufficient quantities for research
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purposes, while T. borchii mycorrhizae have been developed
in vitro (Sisti et al., 1998). Pure cultures of other Tuber species
have been obtained, opening up the possibility of extending
the applications of truffle research (Iotti et al., 2002).
Notwithstanding the progress in truffle research, many
questions are still fully open as far as truffles in truffle-
grounds are concerned: (i) How abundant are truffle
mycorrhizae in a natural truffle-ground? (ii) Can we detect
mycorrhizae exclusively in productive zones, or also in
nonproductive ones? (iii) What other fungi are present in a
truffle-ground? These questions stimulated researchers to
investigate the distribution of the symbiotic phase of
T. magnatum in a selected truffle-ground where the produc-
tion of fruiting bodies had been followed for as long as 5 years
(Mello et al., 2005). This attempt was essential to know the
ecological behaviour of this fungus which cannot be obtained
either as a pure culture or from artificial truffle-grounds.
The simplest method to investigate a fungal species
during its symbiotic phase is the morphological typing of
mycorrhizae in nature. This method, however, is dependent
on many factors, such as age, host tree species and environ-
mental conditions. For this reason, morphological typing of
ectomycorrhizae is currently supported by molecular meth-
ods. In the screening of mycorrhizal tips in the selected
truffle-ground, T. magnatum mycorrhizae were found to be
very rare: it seems that this fungus invests more in fruiting
body formation than in colonization of the roots (Murat
et al., 2005). Similar results have also been obtained in
another T. magnatum area in central Italy (Bertini et al.,
2006). Furthermore, T. magnatum mycorrhizae were present
in a nonproductive period for T. magnatum, and in a
nonproductive area of the truffle-ground, indicating that
there is not a direct linkage between mycorrhizae and
fruiting bodies (Murat et al., 2005). All these observations
raise questions about the functional role of the truffle’s
symbiotic phase, suggesting that truffles may be more plastic
in their metabolism than expected. They seem to move
among different nutritional strategies (saprobic, endophytic
and symbiotic) depending on the environment and on the
developmental phase of their life cycle. On this issue, a
recent report shows that some Tuber species can also be
c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
6 A. Mello et al.
endophytic inside roots of chlorophyllous and achlorophyl-
lous plants such as orchids, suggesting a possible role as a
bridge between mycoheterotrohic species and ectomycor-
rhizal trees (Selosse et al., 2004).
As truffle fruiting bodies are hypogeous it is likely that soil
micro-fauna and microorganisms could enhance or inhibit
truffle formation (Callot et al., 1999). Many studies have
shown that numerous soil microorganisms interact with
fungi promoting antagonistic, competitive or synergistic
activities (Garbaye & Bowen, 1989; Frey-Klett et al., 1999).
Only a few studies have focused on the fungal biodiversity
in truffle-grounds. Luppi-Mosca (1973) identified some
fungi which seem common to the truffle environment. In a
study of three T. aestivum Italian truffle-grounds, Zacchi
et al. (2003) isolated several yeast species, among which,
Cryptococcus strains appear to be specific to this habitat.
From the activities shown, Cryptococcus humicolus, present
on the surface of mature truffles, may contribute to Tuber
nutrition during the saprotrophic stage or facilitate fungal
ascospore dispersion. Furthermore, Buzzini et al. (2005)
found that yeast isolates from T. magnatum and T. melanos-
porum ascocarps produced some molecules characteristic of
the complex aroma of truffles. This suggested that yeasts
have a complementary role in contributing to the final Tuber
aroma. From these results, it is clear that this role needs to be
explored more deeply. Murat et al. (2005), using both
morphological and molecular approaches, found that The-
lephoraceae, Pezizales and Sebacinaceae were the dominant
fungal taxa in the subterranean ECM community in a T.
magnatum truffle-ground. While this study provided a
snapshot of the fungal community, more studies are surely
needed to completely describe both the fungal biodiversity
and the interactions with truffles.
In order to characterize the bacterial populations of
mycorrhizal fruiting bodies, many studies based on cultiva-
tion methods have been performed, leading to the conclu-
sion that Pseudomonas, aerobic spore-forming bacteria and
actinomycetes are the most frequently isolated organisms
within ascoma or ectomycorrhiza of Tuber spp. (Bedini
et al., 1999; Sbrana et al., 2002). Gazzanelli et al. (1999) also
assigned to these organisms a potential role in the symbiosis,
assuming that certain pseudomonads and bacilli, which
express a clear chitinolitic activity, are able to affect ascos-
pore germination within fruiting bodies of T. borchii facil-
itating ascus opening. Furthermore, the finding for
Cytophaga–Flexibacter 16S rRNA gene sequences in T.
borchii ascocarps, mycelium and ectomycorrhizae, provides
the first evidence that this bacterium could be involved in
the entire life cycle of T. borchii (Barbieri et al., 2005).
In conclusion, we can be sure that the microbial biodi-
versity of truffle-grounds affects their productivity but, at
the same time, we are far from knowing the mechanisms
involved in this activity. In times of ‘metagenomics’, which is
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
the recovery and analysis by sequencing of the collective
genomes of microorganisms in an environment or niche
(Daniel, 2005), it will be necessary to compare soil samples
coming from areas of different productivity and to highlight
differences in the presence of microorganisms.
Conclusions
The increasing attention towards the highly appreciated and
commercialized hypogeous ascocarps has led to the point
that publications on truffles exceed 1500, so far (Ceruti
et al., 2003). The advent of molecular biology techniques as
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an addition to classical morphology has allowed the devel-
opment of probes able to identify many truffle species along
their life cycle. A website, created and maintained by the
Italian Tuber scientific network (http://www.truffle.org),
hosts detailed information on various Tuber species together
with molecular tools for their identification.
Thanks to polymorphic sites, a reconstruction of the past
history of T. melanosporum and T. magnatum has been
traced. Their postglacial recolonization from the refuges,
resembling that of the host plants, the oaks, is probably the
reason for their present geographic distribution.
At the moment we have only patchy information on
truffles, as we are not able to answer general questions, such
as: how a fruiting body develops from a hyphal net. As
truffle fruiting bodies cannot yet be obtained under con-
trolled conditions, our knowledge of the morphogenetic
events leading to ascocarp development, is quite limited
even if many data are already available (Lacourt et al., 2002;
Zeppa et al., 2002). Regarding this, Gabella et al. (2005)
clearly demonstrated the presence of an active metabolism
in T. borchii fruiting bodies.
The life cycle of truffles is still obscure and the reason for
this can be traced to many factors. On the one hand, absence
of an experimental system based on spore germination has
never allowed the classical breeding of the resulting mycelia.
On the other hand, mating-type genes, which determine
heterothallism and pseudohomothallism, have never been
reported for truffles, unlike for other filamentous Ascomy-
cetes. The T. melanosporum genome sequencing recently
launched by Francis Martin (France, pers. commun.) will
surely help in decoding the mystery of this fascinating
product of nature and enigma for science.
Acknowledgements
We wish to thank F. Martin (INRA) and S. Ottonello
(University of Parma) for their long standing collaboration
in common truffle projects as well as many Italian research-
ers involved in the Strategic Program ‘Biotecnologia dei
funghi eduli ectomicorrizici’ funded by the National Coun-
cil of Research. Our research was supported by ‘Commessa
FEMS Microbiol Lett 260 (2006) 1–8
Truffles: much more than a prized and local fungal delicacy
Biodiversità’-CNR and CEBIOVEM (DM 17/10/2003, n
193/2003) to P.B.
References
Amicucci A, Zambonelli A, Giomaro G, Potenza L & Stocchi V
(1998) Identification of ectomycorrhizal fungi of the genus
Tuber by species-specific ITS primers. Mol Ecol 7: 273–277.
Amicucci A, Zambonelli A, Guidi C & Stocchi V (2001) Morpho-
logical and molecular characterisation of Pulvinula constellatio
ectomycorrhizae. FEMS Microbiol Lett 194: 121–125.
Barbieri E, Bertini L, Rossi I, et al. (2005) New evidence for
bacterial diversity in the ascoma of the ectomycorrhizal fungus
Tuber borchii Vittad. FEMS Microbiol Lett 247: 23–33.
Bedini S, Bagnoli G, Sbrana C, et al. (1999) Pseudomonads
isolated from within fruit bodies of T. borchii are capable of
producing biological control or phytostimulatory compounds
in pure culture. Symbiosis 26: 223–236.
Bellesia F, Pinetti A, Bianchi A & Tirillini B (1998) The volatile
organic compounds of black truffle (Tuber melanosporum Vitt)
from Middle Italy. Flavour Fragr J 13: 56–58.
Bennet KD, Tzedakis PC & Willis KJ (1991) Quaternary refugia of
North European trees. J Biogeogr 18: 103–115.
Bertault G, Raymond M, Berthomieu A, Callot G & Fernandez D
(1998) Trifling variation in truffles. Nature 394: 734.
Bertault G, Rousset F, Fernandez D, Berthomieu A, Hochberg
ME, Callot G & Raymond M (2001) Population genetics and
dynamics of the black truffle in a man-made truffle field.
Heredity 86: 451–458.
Bertini L, Rossi I, Zambonelli A, Amicucci A, Sacchi A, Cecchini
M, Gregori G & Stocchi V (2006) Molecular identification of
Tuber magnatum ectomycorrhizae in the field. Microbiol Res
161: 59–64.
Brillat-Savarin A (1826) Physiologie du Goût, Sautelet, Paris.
Buzzini P, Gasparetti C, Turchetti B, et al. (2005) Production of
volatile organic compounds (VOCs) by yeasts isolated from
the ascocarps of black (Tuber melanosporum Vitt.) and white
(Tuber magnatum Pico) truffles. Arch Microbiol 184: 187–193.
Callot G, Byé P, Raymond M, Fernandez D, Pargney JC, Parguey-
Leduc A, Janex-Favre MC, Moussa R & Pagès L (1999)
La truffe, la terre, la vie, INRA, Paris, 210 pp.
Ceruti A, Fontana A & Nosenzo C (2003) Le specie europee del
genere Tuber, Una revisione storica, Regione Piemonte, Torino.
Chatin AD (1869) La truffe, Bouchard-Huzard, Paris, 202 pp.
Chevalier G (1994) Evolution des recherches sur les plants
mycorhizés par la truffe et perspectives de développement.
Giorn Bot Ital 128: 7–18.
Ciccarelli A (1564) Opusculum de Tuberibus. Pavia.
Daniel R (2005) The metagenomics of soil. Nat Rev Microbiol 3:
470–478.
Douet JP, Castroviejo M, Mabru D, Chevalier G, Dupré C,
Bergougnoux F, Ricard JM & Médina B (2004) Rapid
molecular typing of Tuber melanosporum, T. brumale and T.
indicum from tree seedlings and canned truffles. Anal Bioanal
Chem 379: 668–673.
FEMS Microbiol Lett 260 (2006) 1–8
7
Dupré C, Chevalier G & Branlard G (1985) Caractérisation des
mycorhizes de différents Tuber par l’étude du polymorphisme
enzymatique. C.R. 1er Coll. Natl. Sur les techniques de
purification des protéines, Paris, 1–3 octobre 1984, DIPCINPL
public, pp. 465–467.
Fontana A & Giovannetti G (1978–79) Simbiosi micorrizica fra
Cistus incanus L. spp. incanus e Tuber melanosporum Vitt.
Allionia 23: 5–11.
Frey-Klett P, Churin JL, Pierrat JC & Garbaye J (1999) Dose effect
in the dual inoculation of an ectomycorrhizal fungus and a
mycorrhiza helper bacterium in two forest nurseries. Soil Biol
Biochem 31: 1555–1562.
Downloaded from https://academic.oup.com/femsle/article/260/1/1/539505 by guest on 03 February 2022
Frizzi G, Lalli G, Miranda M & Pacioni G (2001) Intraspecific
isoenzyme variability in Italian population of the white truffle
Tuber magnatum. Mycol Res 105: 365–369.
Gabella S, Abbà S, Duplessis S, Montanini B, Martin F & Bonfante
P (2005) Transcript profiling reveals novel marker genes
involved in fruiting body formation in Tuber borchii.
Eukaryotic Cell 4: 1599–1602.
Gandeboeuf D, Dupré C & Chevalier G (1994) Différenciation
des truffes européennes d’intérêt commercial par analyse des
isoenzymes. Acta Bot Gallica 141: 455–463.
Gandeboeuf D, Dupré C, Roeckel-Drevet P, Nicolas P & Chevalier
G (1997) Grouping and identification of Tuber species using
RAPD markers. Can J Bot 75: 36–45.
Garbaye J & Bowen GD (1989) Stimulation of ectomycorrhizal
infection of Pinus radiata by some microorganisms associated
with the mantle of ectomycorrhizas. New Phytol 112: 383–388.
Gazzanelli G, Malatesta M, Pianetti A, Baffone W, Stocchi V &
Citterio B (1999) Bacteria associated to fruit bodies of the
ecto-mycorrhizal fungus Tuber borchii Vittad. Symbiosis 26:
211–222.
Gevers D, Cohan FM, Lawrence JG, et al. (2005) Opinion: re-
evaluating prokaryotic species. Nat Rev Microbiol 3: 733–739.
Gioacchini AM, Menotta M, Bertini L, Rossi I, Zeppa S,
Zambonelli A, Piccoli G & Stocchi V (2005) Solid-phase
microextraction gas chromatography/mass spectrometry: a
new method for species identification of truffles. Rapid
Commun Mass Spectrom 19: 2365–2370.
Hall IR, Yun W & Amicucci A (2003) Cultivation of edible
ectomycorrhizal mushrooms. Trends Biotechnol 21: 433–438.
Harley JL & Smith SE (1983) Mycorrhizal Symbiosis, Academic
Press, London.
Iotti M, Amicucci A, Stocchi V & Zambonelli A (2002)
Morphological and molecular characterization of mycelia of
some Tuber species in pure culture. New Phytol 155: 499–505.
Kirk PM, David JC & Stalpers JA (2001) Ainsworth and Bisby’s
Dictionary of the Fungi, CAB, Wallingford.
Lacourt I, Duplessis S, Abbà S, Bonfante P & Martin F (2002)
Isolation and characterization of differentially expressed genes
in the mycelium and fruit body of Tuber borchii. Appl Environ
Microbiol 68: 4574–4582.
Lanfranco L, Wyss P, Marzachi C & Bonfante P (1993) DNA
probes for identification of the ectomycorrhizal fungus Tuber
magnatum Pico. FEMS Microbiol Lett 114: 245–225.
c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
8 A. Mello et al.
Le Tacon FJ, Garbaye D, Bouchard G, et al. (1988) Field results
from ectomycorrhizal inoculation in France. Canadian
Workshop on Mycorrhizae in Forestry (Lalonde & Piché, eds),
pp. 51–74. Université Laval, Canada.
Longato S & Bonfante P (1997) Molecular identification of
mycorrhizal fungi by direct amplification of microsatellite
regions. Mycol Res 101: 425–432.
Luppi-Mosca AM (1973) La microflora della rizosfera nelle
tartufaie. Allionia 19: 29–32.
Mabru D, Douet JP, Mouton A, et al. (2004) PCR-RFLP using a
SNP on the mitochondrial LSU-rDNA as an easy method
to differentiate Tuber melanosporum (Perigord truffle)
and other truffle species in cans. Int J Food Microbiol 94:
33–42.
Mello A, Garnero L & Bonfante P (1999) Specific PCR-primers as
a reliable tool for the detection of white truffles in mycorrhizal
roots. New Phytol 141: 511–516.
Mello A, Vizzini A, Longato S, Rollo F, Bonfante P & Trappe JM
(2000) Tuber borchii versus T. maculatum: neotype studies and
DNA analyses. Mycologia 92: 326–333.
Mello A, Fontana A, Meotto F, Comandini O & Bonfante P
(2001) Molecular and morphological characterization of
T. magnatum mycorrhizae in a long-term survey. Microbiol Res
155: 279–284.
Mello A, Cantisani A, Vizzini A & Bonfante P (2002) Genetic
variation of Tuber uncinatum and its relatedness to other black
truffles. Environ Microbiol 4: 584–594.
Mello A, Murat C, Gavazza V, Vizzini A & Bonfante P (2005)
Tuber magnatum Pico, a species of limited geographic
distribution: its genetic diversity inside and outside a truffle-
ground. Environ Microbiol 7: 55–65.
Mouches C, Duthil P, Poitou N, Delmas J & Bove JM (1981)
Caractérisation des espèces truffières par analyse de leurs
protéines en gels de polyacrylamide et application de ces
techniques à la taxonomie des champignons. Mushroom Sci
11: 819–831.
Murat C, Diez J, Luis P, et al. (2004) Polymorphism at the
ribosomal DNA ITS and its relation to postglacial
re-colonization routes of the Perigord truffle Tuber
melanosporum. New Phytol 164: 401–411.
Murat C, Vizzini A, Bonfante P & Mello A (2005) Morphological
and molecular typing of the below-ground fungal community
in a natural Tuber magnatum truffle-ground. FEMS Microbiol
Lett 245: 307–313.
O’Donnell K, Cigelnik E, Weber NS & Trappe JM (1997)
Phylogenetic relationships among ascomycetous truffles and
the true and false morels inferred from 18S and 28S ribosomal
DNA sequence analysis. Mycologia 89: 48–65.
Pacioni G & Pomponi G (1991) Genotypic patterns of some
Italian species of Tuber aestivum–Tuber mesentericum complex.
Mycotaxon 42: 171–179.
Paolocci F, Rubini A, Riccioni C, Topini F & Arcioni S (2004)
Tuber aestivum and Tuber uncinatum: two morphotypes or two
species? FEMS Microbiol Lett 235: 109–115.
c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
Percudani R, Trevisi A, Zambonelli A & Ottonello S (1999)
Molecular phylogeny of truffles (Pezizales: Terfeziaceae,
Tuberaceae) derived from nuclear rDNA sequence analysis.
Mol Phylogenet Evol 13: 169–180.
Petit RJ, Brewer S, Bordacs S, et al. (2002) Identification of refugia
and post-glacial colonisation routes of European white oaks
based on chloroplast DNA and fossil pollen evidence. Forest
Ecol Manage 156: 49–74.
Riousset L, Riousset G, Chevalier G & Bardet MC (2001) Truffes
d’Europe et de Chine, INRA, Paris, 181 pp.
Rubini A, Paolocci F, Granetti B & Arcioni S (1998) Single step
molecular characterization of morphologically similar black
Downloaded from https://academic.oup.com/femsle/article/260/1/1/539505 by guest on 03 February 2022
truffle species. FEMS Microbiol Lett 164: 7–12.
Rubini A, Paolocci F, Granetti B & Arcioni S (2001)
Morphological characterization of molecular-typed Tuber
magnatum ectomycorrhizae. Mycorrhiza 11: 179–185.
Rubini A, Paolocci F, Riccioni C, Vendramin G & Arcioni S
(2005) Genetic and phylogeographic structures of the
symbiotic fungus Tuber magnatum. Appl Environ Microbiol
71: 6584–6589.
Sbrana C, Agnolucci M, Bedini S, Lepera A, Toffanin A,
Giovannetti M & Nuti MP (2002) Diversity of culturable
bacterial populations associated to Tuber borchii
ectomycorrhizas and their activity on T. borchii mycelial
growth. FEMS Microbiol Lett 211: 195–201.
Selosse MA, Faccio A, Scappaticci G & Bonfante P (2004)
Chlorophyllous and achlorophyllous specimens of Epipactis
microphylla (Neottieae, Orchidaceae) are associated with
ectomycorrhizal septomycetes, including truffles. Microbiol
Ecol 47: 416–442.
Sisti D, Zambonelli A, Giomaro G, et al. (1998) In vitro
mycorrhizal synthesis of micropropagated Tilia platyphyllos
Scop. plantlets with Tuber borchii Vittad mycelium in pure
culture. Acta Horticul 457: 379–387.
Stocchi V (1999) Metodi Molecolari per l’Identificazione delle
Diverse Specie di Tartufo. Urbino University, 110 pp.
Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett
DS & Fisher MC (1999) Phylogenetic species recognition and
species concepts in fungi. Fungal Genet Biol 31: 21–32.
Urbanelli S, Sallicandro P, De Vito E, Bullini L, Palenzona M &
Ferrara AM (1998) Identification of Tuber mycorrhizae using
multilocus electrophoresis. Mycologia 90: 389–395.
Weden C, Danell E & Tibell L (2005) Species recognition in the
truffle genus Tuber – the synonyms Tuber aestivum and Tuber
uncinatum. Environ Microbiol 7: 1535–1546.
Zacchi L, Vaughan-Martini A & Angelini P (2003) Yeast distribution
in a truffle-field ecosystem. Ann Microbiol 53: 275–282.
Zeppa S, Guidi C, Zambonelli A, Potenza L, Vallorani L, Pierleoni
R, Sacconi C & Stocchi V (2002) Identification of putative
genes involved in the development of Tuber borchii fruit body
by mRNA differential display in agarose gel. Curr Genet 42:
161–168.
Zhang L, Yang ZL & Song DS (2005) A phylogenetic study of
commercial Chinese truffles and their allies: taxonomic
implications. FEMS Microbiol Lett 245: 85–92.
FEMS Microbiol Lett 260 (2006) 1–8