Abstract

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 Vittadet al, 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.

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 ascospores (Fig. 1b).

1

Tuber magnatum mycorrhizae (a) and ascospores (b).

1

Tuber magnatum mycorrhizae (a) and ascospores (b).

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 (Kirket al,2001). While in traditional classification systems the true truffles were included in the order Tuberales, together with 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'Donnellet al,1997; Percudaniet al,1999).

Some species, such as Tuber magnatum Pico, the ‘white truffle’, and Tuber melanosporum Vittadet al, 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 (Bellesiaet al,1998; Gioacchiniet al,2005). Several biotic (fungi, yeasts, bacteria, mesofauna, plant host) and abiotic (soil composition, weather such as rain, sunshine and temperature) factors could influence truffle life and enhance or inhibit ascocarp formation (Cerutiet 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 (Rioussetet 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 East Europe – Croatia, Slovenia and Hungary – resulting in a limited availability.

At 300–400 Euros per 100g 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.get al., 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 molecular 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 phylogeography, and morphogenesis, have been addressed recently by researchers, taking advantage of the most advanced techniques 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 (Cerutiet al,2003).

Truffle fruiting bodies are usually identified from the size and shape of their spores and asci, spore wall ornamentation, 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. hiemalbum (Rioussetet al,2001).

Biochemical tools, such as one-dimensional gel electrophoresis with total protein (Moucheset al,1981; Dupré, 1985) and isoenzyme analysis (Pacioni & Pomponi, 1991; Gandeboeufet al,1994; Urbanelliet al,1998), were the first to be used to verify the morphological identification of truffles. However, these techniques present several problems: 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), respectively, identified six (Lanfrancoet 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 developed to discriminate T. magnatum from the so-called ‘whitish’T. borchii and T. maculatum characterized by less taste and a lower commercial value (Amicucciet al,1998; Melloet al,2000) (Fig. 2). As well as for white truffles, specific primers distinguish T. melanosporum from T. brumale and the Asiatic Tuber indicum, both of which are used in food in place of T. melanosporum (Rubiniet al,1998; Douetet al,2004). Processed foods sold as containing T. melanosporum can be contaminated with up to 5% of other Tuber species (Mabruet al,2004).

2

 Internal transcribed spacer regions amplification with specific primers P7/M3 from fruitbody and mycorrhizae of Tuber magnatum and from different Tuber spp. Only T. magnatum shows a 434bp band. First lane: molecular size marker pUC18 DNA HaeIII digest. (Modified from Fig. 2 in Melloet al,1999.)

2

 Internal transcribed spacer regions amplification with specific primers P7/M3 from fruitbody and mycorrhizae of Tuber magnatum and from different Tuber spp. Only T. magnatum shows a 434bp band. First lane: molecular size marker pUC18 DNA HaeIII digest. (Modified from Fig. 2 in Melloet al,1999.)

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 diagnostic tools to confirm the occurrence of the desired fungus in the mycorrhizal roots (Stocchi, 1999; Melloet al,2001; Rubiniet 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 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 (Moucheset al,1981). After two publications leading to controversial results (Melloet al,2002; Paolocciet 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 diagnostic, and it is not possible to separate T. aestivum from T. uncinatum (Wedenet 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 (2005). As for prokaryotes, where assignment of isolates to species is based on phenotypic measures and genome similarity (Geverset al,2005), similar difficulties exist for defining the species of fungi (Tayloret 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 questions 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 (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 (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 distances (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 contrast with previous data (Bertaultet al,1998, 2001), it is suggested that genetic determinants play a role in the organoleptic differences of the black truffle.

Bertault (1998) explained the low level of polymorphism 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 (Bennetet 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. melanosporum 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 (Petitet al,2002): the Rhone valley route and the Atlantic route (Muratet al,2004, Fig. 3).

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 (Petitet 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 Muratet al,2004).

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 (Petitet 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 Muratet al,2004).

Like T. melanosporum, T. magnatum has been the subject of studies focused on genetic variability (Lanfrancoet al,1993; Gandeboeufet al,1997; Frizziet al,2001). Thanks to the finding of a single nucleotide polymorphism in T. magnatum ascocarps, it was possible to identify two haplotypes in a northern Italian population and to trace their spatial and temporal distribution (Melloet al,2005, Fig. 4).

4

 Three haplotypes of Tuber magnatum originated by two single nucleotide polymorphisms, indicated by a star in an oligonucleotide sequence (the different colours indicate nucleotides: T, red; C, blue; G, black; A, green). Two haplotypes are present in the northern Italian population (adapted from Fig. 2 in Melloet al,2005).

4

 Three haplotypes of Tuber magnatum originated by two single nucleotide polymorphisms, indicated by a star in an oligonucleotide sequence (the different colours indicate nucleotides: T, red; C, blue; G, black; A, green). Two haplotypes are present in the northern Italian population (adapted from Fig. 2 in Melloet al,2005).

Moreover, analysis of samples coming from Italian and Balkan populations suggested a genetic differentiation in T. magnatum over its habitat (Melloet al,2005), a finding which was further confirmed by Rubini (2005) after an extensive analysis. By polymorphic microsatellites, these authors found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations and probably spread from a refuge of T. magnatum in central Italy during the postglacial expansion.

Unlike the precious black and white truffles, other truffle species show a higher genetic diversity, i.e. T. borchii, T. aestivum and T. maculatum (Lanfrancoet al,1993; Gandeboeufet al,1997; Melloet al,2002; Paolocciet al,2004). It is interesting to note that these species have a larger geographical distribution than T. magnatum and T. melanosporum (Rioussetet al,2001); for example, T. maculatum is found from France to Russia. It is probable that quaternary 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. Zambonelli 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, reported 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 (Callotet al,1999).

Research programmes for large-scale mycorrhizal production have been elaborated in southern European countries to increase truffle production (Chevalier, 1994). Their main steps are: production of mycorrhizal roots in controlled 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 Taconet 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. melanosporum is not naturally found, such as Israel, the USA and New Zealand (Cerutiet al,2003; Hallet al,2003). At present, T. melanosporum, T. borchii and T. aestivum are collected from artificial truffle-grounds. By contrast, production of T. magnatum in controlled conditions has been rarely reported (and it is not certain whether it depends on 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 sppet al, 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 (Amicucciet al,2001). However, only T. borchii mycelium has been produced in sufficient quantities for research purposes, while T. borchii mycorrhizae have been developed in vitro (Sistiet al,1998). Pure cultures of other Tuber species have been obtained, opening up the possibility of extending the applications of truffle research (Iottiet 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 production of fruiting bodies had been followed for as long as 5 years (Melloet 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 environmental conditions. For this reason, morphological typing of ectomycorrhizae is currently supported by molecular methods. 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 (Muratet al,2005). Similar results have also been obtained in another T. magnatum area in central Italy (Bertiniet 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 alet 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 endophytic inside roots of chlorophyllous and achlorophyllous plants such as orchids, suggesting a possible role as a bridge between mycoheterotrohic species and ectomycorrhizal trees (Selosseet al,2004).

As truffle fruiting bodies are hypogeous it is likely that soil micro-fauna and microorganisms could enhance or inhibit truffle formation (Callotet al,1999). Many studies have shown that numerous soil microorganisms interact with fungi promoting antagonistic, competitive or synergistic activities (Garbaye & Bowen, 1989; Frey-Klettet 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 (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 (2005) found that yeast isolates from T. magnatum and T. melanosporum 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 (2005), using both morphological and molecular approaches, found that Thelephoraceae, 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 cultivation methods have been performed, leading to the conclusion that Pseudomonas, aerobic spore-forming bacteria and actinomycetes are the most frequently isolated organisms within ascoma or ectomycorrhiza of Tuber spp. (Bediniet al,1999; Sbranaet al,2002). Gazzanelli (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 ascospore germination within fruiting bodies of T. borchii facilitating 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 (Barbieriet al,2005).

In conclusion, we can be sure that the microbial biodiversity 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 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 (Cerutiet al,2003). The advent of molecular biology techniques as an addition to classical morphology has allowed the development 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 controlled conditions, our knowledge of the morphogenetic events leading to ascocarp development, is quite limited even if many data are already available (Lacourtet al,2002; Zeppaet al,2002). Regarding this, Gabella (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 Ascomycetes. 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 researchers involved in the Strategic Program ‘Biotecnologia dei funghi eduli ectomicorrizici’ funded by the National Council of Research. Our research was supported by ‘Commessa 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
)
Morphological 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
.
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
1979)
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
.
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
.
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
), 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 aestivumTuber 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
.
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 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
.