10.6092/issn.2465-311X/5593
I tartufi sono funghi ascomiceti appartenenti all’ordine delle Pezizales anche se molti ricercatori considerano “veri tartufi” solo le specie apparteneti al genere Tuber, che comprende le specie di maggiore interesse gastronomico e commerciale quali Tuber melanosporum (tartufo nero pregiato), Tuber magnatum (tartufo bianco pregiato), Tuber aestivum (tartufo estivo o uncinato) e Tuber borchii (tartufo bianchetto). L’elevato valore economico di questi tartufi ha suscitato grande interesse riguardo la loro coltivazione fin dal lontano rinascimento. La nascita della tartuficoltura moderna, tuttavia, risale agli anni 70 in seguito alla scoperta della naura ectomicorrizica del tartufo. Oggi la tartuficoltura è diventata un’importante attività agricola in Europa e nel mondo. In Europa la produzione di T. melanosporum nelle tartufaie coltivate supera addirittura quella delle tartufaie naturali, mentre in Australia, dove il tartufo non cresce naturalmente, si ritiene che la produzione nel 2015 abbia raggiunto le 8 tonnellate. Oltre al tartufo nero pregiato sono coltivati con successo anche T. aestivum e T. borchii, mentre la coltivazione del tartufo bianco pregiato è ancora problematica. Comunque, nonostante i significativi successi raggiunti, alcune tartufaie restano improduttive o producono pochi ascomi. Ciò è conseguenza anche della mancanza di un comune regolamento europeo di certificazione delle piantine tartufigene che garantisca ai tartuficoltori la presenza di un prodotto vivaistico di qualità in tutti i mercati europei. Attualmente le piantine micorrizate sono prodotte esclusivamente mediante inoculazione sporale; l’applicazione dell’inoculazione miceliare potrebbe migliorare le tecniche di coltivazione, rendendo possibile l’impiego di ceppi geneticamente selezionati per le loro caratteristiche ecologiche e/o produttive. Il sequenziamento del genoma del tartufo nero pregiato (T. melanosporum) ha permesso di chiarire alcune fasi del ciclo biologico del tartufo; in particolare, è stato scoperto che è un fungo eterotallico e, perciò, la produzione dei corpi fruttiferi è subordinata dall’incontro di due ceppi appartenenti a “mating type” (tipo sessuale) diverso. Il sequenziamento del genoma di altre specie di tartufo potrà fornirci altre informazioni sulla loro biologia contribuendo a migliorare le tecniche di coltivazione. La genetica potrà inoltre fornirci indicazioni utili a capire il ruolo dei microrganismi associati al tartufo e magari a suggerirci approcci biotecnologici alternativi per il loro utilizzo in tartuficoltura.
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Current status of truffle cultivation: recent results and future perspectives
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A. Zambonelli, M. Iotti, I. Hall                                     Micologia Italiana vol. 44 (2015) ISSN 2465-311X
DOI: 10.6092/issn.2465-311X/5593



  Current status of truffle cultivation: recent results
  and future perspectives
  ________________________________________________________________________________

                            Alessandra Zambonelli1, Mirco Iotti1, Ian Hall2

  1
      Department of Agricultural Science, Bologna University, viale Fanin 46, 40127 Bologna Italy
  2
      Truffles & Mushrooms (Consulting) Ltd, P.O. Box 268, Dunedin, New Zealand

  Correspondig Author M. Iotti e-mail: mirco.iotti@univaq.it

  Abstract

       In this review the current status of truffle cultivation in Europe and outside Europe is reported. While the
       cultivation of Tuber melanosporum (Périgord black truffle), Tuber aestivum (summer or Burgundy truffle) and
       Tuber borchii (bianchetto truffle) gave good results, only the Italian white truffle (Tuber magnatum), which is
       the most expensive, has yet to be successfully cultivated.
       In future a revolutionary approach to truffle cultivation would be the application of mycelial inoculation
       techniques for producing Tuber infected plants which will allow to select the fungal strains adapted to specific
       climatic, edaphic conditions and hosts. The new insights which will be gained by the extensive Tuber genome
       sequencing programme will also help to improve truffle cultivation techniques.

       Keywords: Tuber melanosporum; Tuber magnatum; Tuber borchii; Tuber aestivum; cultivation; mycelial
       inoculation


  Riassunto

       I tartufi sono funghi ascomiceti appartenenti all’ordine delle Pezizales anche se molti ricercatori considerano
       “veri tartufi” solo le specie apparteneti al genere Tuber, che comprende le specie di maggiore interesse
       gastronomico e commerciale quali Tuber melanosporum (tartufo nero pregiato), Tuber magnatum (tartufo
       bianco pregiato), Tuber aestivum (tartufo estivo o uncinato) e Tuber borchii (tartufo bianchetto). L’elevato
       valore economico di questi tartufi ha suscitato grande interesse riguardo la loro coltivazione fin dal lontano
       rinascimento. La nascita della tartuficoltura moderna, tuttavia, risale agli anni 70 in seguito alla scoperta della
       naura ectomicorrizica del tartufo.
       Oggi la tartuficoltura è diventata un’importante attività agricola in Europa e nel mondo. In Europa la
       produzione di T. melanosporum nelle tartufaie coltivate supera addirittura quella delle tartufaie naturali, mentre
       in Australia, dove il tartufo non cresce naturalmente, si ritiene che la produzione nel 2015 abbia raggiunto le 8
       tonnellate. Oltre al tartufo nero pregiato sono coltivati con successo anche T. aestivum e T. borchii, mentre la
       coltivazione del tartufo bianco pregiato è ancora problematica. Comunque, nonostante i significativi successi
       raggiunti, alcune tartufaie restano improduttive o producono pochi ascomi. Ciò è conseguenza anche della
       mancanza di un comune regolamento europeo di certificazione delle piantine tartufigene che garantisca ai
       tartuficoltori la presenza di un prodotto vivaistico di qualità in tutti i mercati europei. Attualmente le piantine
       micorrizate sono prodotte esclusivamente mediante inoculazione sporale; l’applicazione dell’inoculazione
       miceliare potrebbe migliorare le tecniche di coltivazione, rendendo possibile l’impiego di ceppi geneticamente
       selezionati per le loro caratteristiche ecologiche e/o produttive. Il sequenziamento del genoma del tartufo nero

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A. Zambonelli, M. Iotti, I. Hall                                      Micologia Italiana vol. 44 (2015) ISSN 2465-311X
DOI: 10.6092/issn.2465-311X/5593

       pregiato (T. melanosporum) ha permesso di chiarire alcune fasi del ciclo biologico del tartufo; in particolare, è
       stato scoperto che è un fungo eterotallico e, perciò, la produzione dei corpi fruttiferi è subordinata dall’incontro
       di due ceppi appartenenti a “mating type” (tipo sessuale) diverso. Il sequenziamento del genoma di altre specie
       di tartufo potrà fornirci altre informazioni sulla loro biologia contribuendo a migliorare le tecniche di
       coltivazione. La genetica potrà inoltre fornirci indicazioni utili a capire il ruolo dei microrganismi associati al
       tartufo e magari a suggerirci approcci biotecnologici alternativi per il loro utilizzo in tartuficoltura.

       Parole chiave: Tuber melanosporum, Tuber magnatum, Tuber borchii, Tuber aestivum, coltivazione,
       inoculazione miceliare


  Introduction

  Truffles are ascomyceteous fungi belonging to several families in the Pezizales that have independently
  evolved a subterranean mode of existence (Læssøe & Hansen, 2007). While some basidiomycetes have also
  evolved underground fruiting bodies, in sensu stricto, only species in the genus Tuber, Tuberaceae, are
  considered the true truffles (Jeandroz et al., 2008). The genus has been estimated to contain 180 to 230
  species (Bonito et al., 2010) distributed worldwide. Most species produce strong aromas to attract their
  dispersal agents and a few have considerable economic value because of their unique aromas and flavors. All
  the truffles live in mycorrhizal symbiosis with the roots of suitable host plants (Bonito et al., 2013). Tuber
  were thought to only form ectomycorrhizas but recently it was found that they are also able to form arbutoid
  mycorrhizas (Lancellotti et al., 2014) and endomycorrhizas with orchids (Selosse et al., 2004).




                Fig. 1 - Ascomata of T. magnatum (a), T. borchii (b), T. melanosporum (c) and T. aestivum (d)
                Fig. 1 - Ascomi di T. magnatum (a), T. borchii (b), T. melanosporum (c) e T. aestivum (d)

  The most valuable truffles are the European species Tuber melanosporum Vittad. (Périgord black truffle),
  Tuber magnatum Pico (Italian white truffle), Tuber aestivum Vittad. (summer or Burgundy truffle) and
  Tuber borchii Vittad. (bianchetto truffle) (Fig. 1) (Hall et al., 2007). Other species with a limited market in
  Europe are Tuber brumale, Tuber macrosporum Vittad. and Tuber mesentericum Vittad. In the U.S.A.
  several species of truffle are harvested but only the Oregon white truffles (Tuber oregonense and Tuber
  gibbosum) and the pecan truffle (Tuber lyonii) have a limited local market (Trappe, 2009; Lefevre, 2012).
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DOI: 10.6092/issn.2465-311X/5593


  There are many species of truffle in China. Some resemble European species and some are collected and
  exported to Europe. The Chinese black truffle, Tuber indicum Cooke & Massee, is quite similar to T.
  melanosporum but with a relatively poor flavor (Riousset et al., 2001) and Tuber sinoaestivum which is
  similar to the European T. aestivum (Zambonelli et al., 2012; Zhang et al., 2012). Tuber oligosperum (Tul. &
  Tul.) Trappe is harvested in North Africa, in particular Morocco, and illegally sold in Italy as T. magnatum
  (Boutahir et al., 2013).
  The high economic value of truffles has stimulated researchers to find the most efficient methods for
  cultivating them. This has not been a simple task because of their complex life cycle involves a symbiotic
  relationship with suitable host trees and it is entirely completed underground where complex relationships
  with soil microorganisms are involved.
  Here, we briefly report on the current status of truffle cultivation, the most recent research information and
  the future prospects for these diamonds of cuisine.

  Early methods

  The first Italian and French attempts at cultivating truffles date back to the Renaissance, but successful and
  commercial cultivation of truffles did not start until the early 1800s when Josef Talon in France developed a
  crude but effective method for cultivating the Périgord black truffle (Hall et al., 2007). This technique
  involved sowing acorns collected under oaks that were producing truffles. It was very successful in France in
  areas where truffle spores were already present in soil but when applied in Italy the method failed (Mannozzi
  Torini, 1984). Modern truffle cultivation was introduced into France and Italy in the 1970’s after the
  discovery of the mycorrhizal nature of truffles.
  Initially three methods for inoculating plants were tried: spore inoculation, mother plant technique and
  mycelial inoculation. The mother plant technique involved planting seedlings into the rooting zone of a plant
  know to be mycorrhized with the required truffle. While mycelial inocula also showed promise (Chevalier,
  1973) spore inoculation soon became the method of choice for the commercial production of plants.

  Current status of truffle cultivation

  In the second middle of 1900s truffle cultivation became popular had a great impulse not only in Europe but
  also in non-European countries. T. melanosporum is the truffle which has been the most successfully
  cultivated around the world but the cultivation of T. aestivum and T. borchii has also been successful in
  many countries.
  T. melanosporum cultivation
  Most of the black truffles produced in Italy, France and Spain (Fig. 2) are now harvested from cultivated
  truffières and in France only 10% are now harvested from natural areas (Reyna & Garcia-Barreda, 2014).
  Truffle cultivation gave also very good results in countries like New Zealand, USA and Australia where
  truffles were introduced by cultivation. In these countries most of the soils are acidic and in their native state
  unsuited for truffle cultivation. However, Ian Hall showed that some soils could be modified first by
  intensive liming and then correcting the almost inevitable trace element deficiencies (Hall et al., 2007). This
  method soon spread to Australia which produced 3 tonnes in 2011 (Hall & Haslam, 2012), which is projected
  to grow to 8 tonnes in 2015 (Duell, 2012), and could soon overtake French truffle production at least in a
  poor fruiting year (Hall & Zambonelli, 2012). Cultivated black truffle have also been produced in the USA,
  Canada, Morocco, Chile and South Africa (Reyna & Garcia-Barreda, 2014; Berch & Bonito, 2014;
  Zambonelli unpublished data). T. melanosporum plantations have also been established in Israel and in
  several European countries but to the best of our knowledge production has yet to begin.

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                     Fig. 2 - T. melanosporum plantation in Spain
                     Fig. 2 - Tartufaia coltivata di T. melanosporum in Spagna
  T. magnatum cultivation
  Of the main European truffles only the Italian white truffle, which is the most expensive, has yet to be
  successfully cultivated. Only few truffières have produced in Italy. Despite extensive plantings of inoculated
  trees in Italy from the early 1980s to the late 1990s few truffières have produced and then only a few
  kilograms per hectare (Gregori et al., 2010). The main reason for this seems to be the difficulties in obtaining
  Tuber infected plants in the nursery and significant contamination problems. Before the introduction of
  molecular methods for the identification of the mycorrhizas, T. magnatum inoculated plants were often sold
  contaminated with other less valuable Tuber species such as Tuber maculatum Vittad. and T. borchii. In fact
  these highly infective truffles, which are morphologically similar to T. magnatum, when accidentally
  incorporated in the inoculum colonized the entire root systems (Hall et al., 2007). Another factor limitating
  the cultivation of T. magnatum is that its biology and soil ecology is still a mystery. Its mycorrhizas can be
  obtained in the nursery (Mello et al., 2001; Rubini et al., 2001), albeit with difficulties, but then they seem to
  disappear in the field (Hall et al., 2007). T. magnatum mycorrhizas are also absent in natural productive areas
  (Leonardi et al., 2013). Recent molecular techniques like q-PCR are now able to follow the development of
  T. magnatum mycelium in the soil (Iotti et al., 2012a) and are beginning to give new insights into its soil
  spatio-temporal development (Iotti et al., 2014). The possible role of associate bacteria, and in particular of
  rhizobia, has also been hypothesized in the growth, development, nutrition and fructification of T. magnatum
  (Barbieri et al., 2007; Barbieri et al., 2010). Clearly an in depth study of the strange ecology of this truffle
  will be needed before routine methods for its cultivation can be established.
  T. borchii cultivation
  The first publication reporting the successful cultivation of bianchetto truffle was by Zambonelli and
  colleagues in 2000 in Marina di Ravenna, Italy. Since then its cultivation has become widespread in Italy and
  more recently in New Zealand where this truffle is particularly appreciated by gourmets and in particular the
  Chinese community (Hall et al., 2007; Wong personal communication). The first successful cultivation of T.
  borchii      in     Australia     by     Peter       Stahle     was      reported    in     March       2015
  (https://trufflefarming.wordpress.com/2015/03/14/first-tuber-borchii-ever-harvested-in-australia/).
  Recently, T. borchii cultivation was successfully introduced in USA (Isikhuemhen personal communication).
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  T. borchii cultivation has a great potential because of its broad ecological adaptability, its wide range of
  angiosperm hosts such as oaks, hazel, and linden, and conifers including pines and cedars (Hall et al., 2007;
  Zambonelli et al., 2002). Recently it was found to produce mycorrhizas with the pecan tree Carya
  illinoinensis (Wangenh.) K.Koch, and to fruit when associated with Arbutus unedo L. opening up the
  possibility of combining both the cultivation of this precious truffle and the production of edible fruits of
  these plants (Benucci et al., 2012; Lancellotti et al., 2014). However, there will be difficulties because pecan
  nuts and A. unedo fruits are ready for picking just as the truffles are forming in the soil. Also some rather
  toxic insecticides with residual activity have been used to control pecan pests.
  T. aestivum cultivation
  T. aestivum cultivation in Italy and in France began a little later than the cultivation of T. melanosporum but
  has grown rapidly and is now widespread. In France the cultivation of T. aestivum is concentrated within its
  natural boundaries: Auvergne, Champagne, Lorraine, Bourgogne, and Franche-Comte (Chevalier and
  Frochot, 1997). In Italy out of around 120,000 truffle trees planted per year only 15% are inoculated with T.
  aestivum (Bencivenga et al., 2009). T. aestivum has also been successfully cultivated in Sweden by Christina
  Weden (Weden et al., 2009) on the island of Gotland which represents the northernmost boundary of its
  natural distribution in Europe. Like T. borchii, T. aestivum has a great potential because of its adaption to a
  wide range of soils, climate and host plants. Recent inoculation experiments revealed that mycorrhization of
  pecan tree C. illinoinensis with T. aestivum is also possible which makes truffle–nut coproduction a
  possibility (Benucci et al., 2012).

  The problems

  Despite the successful cultivation of T. melanosporum, T. borchii and T. aestivum there are still some
  significant problems with their cultivation. Some truffières are not productive or produce few truffles of poor
  quality. This is often due to the poor quality of plants produced by some companies. In Europe
  morphological and molecular certification methods for Tuber infected plants are locally applied but an
  European law and a unique certification protocol is needed to protect truffle farmers from failures (Andrés-
  Alpuente et al., 2015; Murat, 2014). What also makes truffle cultivation uncertain are the unpredictable
  genetic characteristics of the truffle strains when plants are inoculated with genetically highly variable
  spores, which may or may not be the most suited to the edaphic and climatic conditions on a particular site.
  The sequencing of the T. melanosporum genome has revealed that it is heterothallic so that strains carrying
  different mating types have to cross for fruiting body production (Martin et al., 2010). Both the mating type
  genes (MAT1-1-1 and MAT1-2-1) were identified and then characterized (Rubini et al., 2011a). After this
  discovery, studies were carried out in order to verify if the lack of productivity in some truffières was due to
  the absence of one of the mating types. However, mating type analyses of seedlings planted in Australia
  showed that both mating types are present suggesting that there are more factors involved in ascocarp
  production than just the presence of both mating types on host trees (Linde & Selmes, 2012). Recent studies
  carried out in Europe showed that even in natural productive areas mating type distribution is patchy and
  often unbalanced (Rubini et al., 2014). In fact, genetically different truffle strains compete with each other
  and with few genets of the same mating type colonize distinct patches of the truffières (Rubini et al., 2011b;
  Murat et al., 2013). Some Authors have hypothesized that such spatial segregation may be due to competitive
  exclusion, with use of the MAT locus as a marker for self-recognition, probably in addition to other
  polymorphic loci (Selosse et al., 2013). The presence of a vegetative incompatibility system associated with
  the mating type genes was demonstrated in Neurospora crassa Shear & B.O. Dodge (Shiu & Glass 1999). In
  this fungal species the tol gene, which is turned off during the sexual cycle, interact with MAT A-1 and
  MAT a-1 to form a heterocomplex that mediates vegetative incompatibility. However, a similar homologous
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  gene has not been found in T. melanosporum (Iotti et al., 2012c). In addition the other het domain genes,
  which in other ascomycetes trigger a programmed cell death after hyphal fusion between incompatible
  strains (Saupe 2000), have not been found in T. melanosporum (Iotti et al., 2012c). Instead hyphal
  anastomoses between strains seem to be prevented by other unknown pre-fusion mechanisms (Iotti et al.,
  2012c) (Fig. 3).
  How sexually compatible strains meet and how fertilization occurs is also still an unknown. The discovery of
  a mitosporic stage in several Tuber species suggests the possible role of the conidia in fertilization process
  like in numerous other ascomycetes (Healy et al., 2012; Carris et al., 2015). Many truffle farmers broadcast
  low quality truffles onto their truffières in the hope of increasing production. Where this does prove fruitful it
  might be through introducing new mating strains. However, this practice comes with risks. Often the farmers
  will spread rotting pieces of truffle which may introduce pathogens. It might also induce a huge genetic load
  for future generations (Selosse et al., 2013). In nature the role of the farmer spreading mating strains might
  also be played by mycophagic animals. These are attracted by truffle aromas, eat the truffles and then spread
  the spores in their feces which remain viable even after passage through the gut (Piattoni et al., 2012;
  Piattoni et al., 2014).

  The future
  A revolutionary approach to truffle cultivation would be the use selected mycelial inoculum adapted to
  specific climatic, edaphic conditions and hosts. This technique was explored at the beginning of modern




  Fig. 3 – Interaction between hyphae of T. melanosporum: A) hyphal anastomosis between hyphae of the same strain,
  arrow indicates the fusion point; B) interaction between hyphae of different strains: no fusion occurs. Bars = 10 µm
  Fig. 3 – Interazione fra ife di T. melanosporum : A) anastomosi ifale fra ife appartenenti allo stesso ceppo, la freccia
  indica il punto di fusione; B) interazione fra ife appartenenti a ceppi diversi: nonostante le ife siano a stretto contatto
  non c’è fusione. Barra = 10 µm



  truffle cultivation by Gerard Chevalier (1973) but it has only been used for research exploring the
  interrelationships the fungi have with their host (Giomaro et al., 2005). One of the biggest limitations to the
  commercial application of this technique will be the difficulty in isolating and maintaining Tuber mycelia in
  pure culture as well as producing mycelial biomass on a large scale (Iotti et al., 2002; Iotti et al., 2012b).
  Moreover long-time subculturing in axenic conditions in the absence of host roots results in the loss of
  isolate infectivity (Boutahir, 2013) and the development of adequate cryopreservation protocols must be used
  to maintain culture viability (Iotti et al., 2012b). In the past another limit in using mycelial inoculants was the

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  lack of knowledge about truffle sexuality resulting in the possibility of producing plants incapable of
  producing truffles. This is because pure cultures of mycelia are isolated from the gleba of fruiting bodies and
  hence only carry the maternal mating type. However, thanks to the characterization of the mating type genes
  of T. melanosporum and of the other edible truffles (Paolocci personal communication) we are now able to
  produce plants inoculated with strains of both mating types.
  Inoculating plants with cultures of Tuber spp. adapted to a specific set of conditions would facilitate the
  selection of better-performing fungal genotypes using the same principles that govern the selection of plant
  cultivars with improved performance. The extensive Tuber genome sequencing program will help to
  determine the genes controlling the qualitative and quantitative characters of truffles but also their
  adaptability to different environmental conditions. Recent studies carried out by Zambonelli’s research group
  has shown that different strains of T. borchii have a different resistance to high temperature (unpublished
  data). This aspect is particularly important when truffle cultivation is introduced in countries having different
  climatic conditions to Europe. It could also help to choose strains having the capacity to adapt to global
  climatic changes. It is also known that plant genotype can also influence truffle production and so some
  nurseries now use clonal plants selected for truffle production (Robin & Cammalletti, 2001).


  Conclusions

  Truffle cultivation is an important agricultural activity in many parts of Europe and is growing in popularity
  worldwide particularly in Southern Hemisphere countries that aim to produce truffles counter season to the
  Northern Hemisphere.
  However problems still exist and need to be addressed. Basic research is clearly needed to better understand
  the biology of truffles and in particular to unravel the mystery around the sexual stages in the lifecycles.
  Studies are also needed to elucidate the mysteries surrounding the soil ecology of T. magnatum in order to
  make its cultivation feasible.
  Future challenges for truffle cultivation also include: adapting modern mycelial inoculation technology to
  large scale production of mycorrhized plants, the creation and identification of elite fungal and plant
  cultivars; maintaining germplasm collections of these cultivars, and the selection of the best cultivars for
  different ecological conditions. The possibility to selectively introducing mycorrhizal helper bacteria and
  perhaps other fungi, which could directly or indirectly affect ectomycorrhizal development and fruit body
  formation, could be another perspective for future truffle cultivation.

  References

  ANDRÉS-ALPUENTE A., SÁNCHEZ S., MARTÍN M., AGUIRRE J., BARRIUSO J. (2014). Comparative analysis of
    different methods for evaluating evergreen oaks mycorrhized with black truffle. Mycorrhiza 24(Suppl 1):
    S29–37.
  BARBIERI E., CECCAROLI P., SALTARELLI R., GUIDI C., POTENZA L., BASAGLIA M., FONTANA F., BALDAN
    E., CASELLA S., RYAHI O., et al. (2010). New evidence for nitrogen fixation within the Italian white
    truffle Tuber magnatum. Fungal Biol 114: 936–942.
  BARBIERI E., GUIDI C., BERTAUX J., FREY-KLETT P., GARBAYE J., CECCAROLI P., SALTARELLI R.,
    ZAMBONELLI A., STOCCHI V. (2007). Occurrence and diversity of bacterial communities in Tuber
    magnatum during truffle maturation. Environ Microbiol 13: 2234–2246.
  BENCIVENGA M., DI MASSIMO G., DONNINI D., BACIARELLI FALINI L. (2009). The cultivation of truffles in
    Italy. Acta Bot Yunnanica suppl XVI: 21–28.



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  BENUCCI G.M.N., BONITO G., BACIARELLI-FALINI L., BENCIVENGA M. (2012). Mycorrhization of Pecan
    trees (Carya illinoinensis) with commercial truffle species: Tuber aestivum Vittad. and Tuber borchii
    Vittad. Mycorrhiza 22(5): 383-392.
  BERCH S.M., BONITO G. (2014). Cultivation of Mediterranean species of Tuber (Tuberaceae) in British
    Columbia, Canada. Mycorrhiza 24(6):473-479.
  BONITO G., SMITH M.E., NOWAK M., HEALY R.A., GUEVARA G., CAZARES E., KINOSHITA A., NOUHRA
    E.R., DOMINGUEZ L.S., et al. (2013). Historical biogeography and diversification of truffles in the
    Tuberaceae and their newly identified southern hemisphere sister lineage. PloS one 8: e52765.
  Bonito G.M., Gryganskyi A.P., Trappe J.M., Vilgalys R. (2010). A global meta-analysis of Tuber ITS rDNA
    sequences: species diversity, host associations and long-distance dispersal. Mol Ecol 19: 4994–5008.
  BOUTAHIR S. (2013). Nuove biotenologie per la produzione di piante micorrizate con tartufo. Dissertation
    thesis, Alma Mater Studiorum Università di Bologna. DOI 10.6092/unibo/amsdottorato/5646.
  BOUTAHIR S., IOTTI M., PIATTONI F., ZAMBONELLI A. (2013). Morphological and molecular characterization
    of Tuber oligospermum mycorrhizas. Afr J Agric Res 8(29): 4081–4087.
  CARRIS L.M., PEEVER T.L., MCCOTTER S.W. (2015). Mitospore stages of Disciotis, Gyromitra and
    Morchella in the inland Pacific Northwest. Mycologia, preliminary version published on April 24, 2015
    as doi:10.3852/14-207.
  CHEVALIER G. (1973). Synthese axenique des mycorhizes de Tuber brumale Vitt. a partir de cultures pures
    du champignon. Annales de Phytopathologie 5(2): 163-182.
  CHEVALIER G., FROCHOT H. (1997). La Truffe de Bourgogne. Editions Pétrarque, Levallois-Perret.
  DUELL G. (2012). The President’s Report. [online]. National Conference of the Australian Truffle Growers
    Association. Available in http://www.trufflegrowers.com.au/wp-content/uploads/2012/09/2012-
    Presidents-Report.pdf. [13 July 2013].
  GIOMARO G., SISTI D., ZAMBONELLI A. (2005). Cultivation of edible ectomycorrhizal fungi by in vitro
     mycorrhizal synthesis. In: In vitro Culture of Mycorrhizas (Declerck S, Strullu DG, Fortin JA eds) Soil
     Biology series, vol.4. Springer-Verlag, Berlin Heidelberg, Germany. pp. 253–267.
  GREGORI G., DONNINI D., BENCIVENGA M. (2010). Tuber magnatum: alcuni esempi produttivi di tartufaie
    coltivate in Italia. In: Atti 3° Congresso Internazionale di Spoleto sul tartufo (Comunità Montana dei
    Monti Martani, Serano e Subasio ed), 25-28 Novembre, Spoleto. pp. 741-749.
  HALL I.R., BROWN G.T., ZAMBONELLI A. (2007). Taming the truffle. Timber Press, Portland.
  HALL I.R., HASLAM W. (2012). Truffle cultivation in the Southern Hemisphere. In: Edible
    ectomycorrhizal mushrooms Current knowledge and future prospects (Zambonelli A, Bonito GM
       eds) Soil Biology Series vol. 34. Springer-Verlag, Berlin, Heidelberg, Germany. pp. 191-208.
  HALL I.R., ZAMBONELLI A. (2012). The Cultivation of Mycorrhizal Mushrooms - Still the Next Frontier! In:
    Mushroom Science XVIII (Zhang j, Wang H, Chen M eds). China Agricultural Press, Beijing, China. pp.
    16-27.
  HEALY R.A., SMITH M.E., BONITO G.M., PFISTER D.H., GE Z.W., GUEVARA G.G., WILLIAMS G., STAFFORD
    K., KUMAR L., LEE T., et al. (2012). High diversity and wide spread occurrence of mitotic spore mats in
    ectomycorrhizal Pezizales. Mol Ecol 22: 1717–1732.
  IOTTI M., AMICUCCI A., STOCCHI V., ZAMBONELLI A. (2002). Morphological and molecular characterisation
     of mycelia of some Tuber species in pure culture. New Phytol 155: 499-505.
  IOTTI M., LEONARDI M., LANCELLOTTI E., SALERNI E., ODDIS M., LEONARDI P., PERINI P., PACIONI G.,
     ZAMBONELLI A. (2014). Spatio-temporal dynamic of Tuber magnatum mycelium in natural truffle
     grounds. PloS one 9(12): e115921.
  IOTTI M., LEONARDI M., ODDIS M., SALERNI E., BARALDI E., ZAMBONELLI A. (2012a). Development and
     validation of a real-time PCR assay for detection and quantification of Tuber magnatum in soil. BMC
     Microbiol 12: 93.
  IOTTI M., PIATTONI F., ZAMBONELLI A. (2012b). Techniques for host plant inoculation with truffles and
     other edible ectomycorrhizal mushrooms. In: Edible ectomycorrhizal mushrooms. Current knowledge and
  38
A. Zambonelli, M. Iotti, I. Hall                             Micologia Italiana vol. 44 (2015) ISSN 2465-311X
DOI: 10.6092/issn.2465-311X/5593

     future prospects (Zambonelli A, Bonito GM eds). Soil Biology Series vol. 34. Springer-Verlag, Berlin,
     Heidelberg, Germany. pp. 145-161.
  IOTTI M., RUBINI A., TISSERANT E., KHOLER A., PAOLOCCI F., ZAMBONELLI A. (2012c). Self/nonself
     recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system. Fungal
     Biol 116: 261-275.
  JEANDROZ S., MURAT C., WANG Y., BONFANTE P., LE TACON F. (2008). Molecular phylogeny and historical
     biogeography of the genus Tuber, the ’true truffles’. J Biogeogr 35: 815–82.
  LÆSSØE T., HANSEN K. (2007). Truffle trouble: what happened to the Tuberales. Mycol Res 111: 1075–
    1099.
  LANCELLOTTI E., IOTTI M., ZAMBONELLI A., FRANCESCHINI A. (2014). Characterization of Tuber borchii
    and Arbutus unedo mycorrhizas. Mycorrhiza 24: 481-486.
  LEFEVRE C. (2012). Native and cultivated truffles of North America. In: Edible ectomycorrhizal mushrooms.
    Current knowledge and future prospects (Zambonelli A, Bonito GM eds). Soil Biology Series vol. 34.
    Springer-Verlag, Berlin, Heidelberg, Germany. pp. 209-226.
  LEONARDI M., IOTTI M., ODDIS M., LALLI G., PACIONI G., LEONARDI P., MACCHERINI S., PERINI C.,
    SALERNI E., ZAMBONELLI A. (2013). Assessment of ectomycorrhizal fungal communities in the natural
    habitats of Tuber magnatum (Ascomycota, Pezizales). Mycorrhiza 23: 349–358.
  LINDE C.C., SELMES H. (2012). Genetic diversity and mating type distribution of Tuber melanosporum and
     their significance to truffle cultivation in artificially planted truffières in Australia. Appl Environ
     Microbiol 78: 6534–6539.
  MANNOZZI TORINI L. (1984).Il tartufo e la sua coltivazione. Edagricole, Bologna.
  MARTIN F., KOHLER A., MURAT C., BALESTRINI R., COUTINHO P.M., JAILLON O., MONTANINI B., MORIN
    E., NOEL B., PERCUDANI R., et al. (2010). Périgord black truffle genome uncovers evolutionary origins
    and mechanisms of symbiosis. Nature 464: 1033-1038.
  Mello A, Fontana A, Meotto F, Comandini O, Bonfante P (2001) Molecular and morphological
    characterization of T. magnatum mycorrhizas in a long-term survey. Microbiol Res 155: 279–284
  MURAT C. (2015). Forty years of inoculating seedlings with truffle fungi: past and future perspectives.
    Mycorrhiza 25: 77–81.
  MURAT C., RUBINI A., RICCIONI C., DE LA VARGA H., AKROUME E., BELFIORI B., GUARAGNO M., LE
    TACON F., ROBIN C., HALKETT F., et al. (2013). Fine-scale spatial genetic structure of the black truffle
    (Tuber melanosporum) investigated with neutral microsatellites and functional mating type genes. New
    Phytol 199: 176–187.
  PIATTONI F., AMICUCCI A., IOTTI M., ORI F., STOCCHI V., ZAMBONELLI A. (2014). Viability and morphology
     of Tuber aestivum spores after passage through the gut of Sus scrofa. Fungal Ecol 9: 52–60.
  PIATTONI F., ORI F., MORARA M., IOTTI M., ZAMBONELLI A. (2012). The role of wild boars in spore
     dispersal of hypogeous fungi. Acta Mycologica 47(2): 145-153.
  REYNA S., GARCIA-BARREDA S. (2014). Black Truffle cultivation: a global reality. For Syst 23(2): 317–328.
  RIOUSSET L., RIOUSSET G., CHEVALIER G. (2001). Truffles d′Europe et de Chine. INRA, Paris.
  ROBIN B., CAMMALLETTI P. (2001). Procuction de plants truffiers d’excellence certifiee ISO 9002 sous
    licence et controle INRA. In: Actes du V Congrès International Science et Culture de la Truffe
    (Federation Francaise des truficulteurs ed). Paris, pp. 320-323.
  RUBINI A., BELFIORI B., RICCIONI C., ARCIONI S., MARTIN F., PAOLOCCI F. (2011b). Tuber melanosporum:
    mating type distribution in a natural plantation and dynamics of strains of different mating types on the
    roots of nursery-inoculated host plants. New Phytol 189: 723–735.
  RUBINI A., BELFIORI B., RICCIONI C., TISSERANT E., MARTIN F., PAOLOCCI F. (2011a). Isolation and
    characterization of MAT genes in the symbiotic ascomycete Tuber melanosporum. New Phytol 189: 710–
    722.


                                                                                                          39
A. Zambonelli, M. Iotti, I. Hall                           Micologia Italiana vol. 44 (2015) ISSN 2465-311X
DOI: 10.6092/issn.2465-311X/5593

  RUBINI A., PAOLOCCI F., GRANETTI B., ARCIONI S. (2001). Morphological characterization of molecular-
    typed Tuber magnatum ectomycorrhizae. Mycorrhiza 11: 179–185.
  RUBINI A., RICCIONI C., BELFIORI B., PAOLOCCI F. (2014). Impact of the competition between mating types
    on the cultivation of Tuber melanosporum: Romeo and Juliet and the matter of space and time.
    Mycorrhiza 24(Suppl 1): S19–S27.
  SAUPE S.J., CLAVE C. BEGUERET J. (2000). Vegetative incompatibility in filamentous fungi: Podospora and
    Neurospora provide some clues. Curr Opin Microbiol 3: 608–612.
  SELOSSE M.A., FACCIO A., SCAPPATICCI G., BONFANTE P. (2004). Chlorophyllous and achlorophyllous
    specimens of Epipactis microphylla (Neottieae, Orchidaceae) are associated with ectomycorrhizal
    septomycetes, including truffles. Microb Ecol 47: 416–42.
  SELOSSE M.A., TASCHEN E., GIRAUD T. (2013). Do black truffles avoid sexual harassment by linking mating
    type and vegetative incompatibility? New Phytol 199: 10–13.
  SHIU P.K.T., GLASS N.L. (1999). Molecular characterization of tol, a mediator of mating-type-associated
     vegetative incompatibility in Neurospora crassa. Genetics 151: 545–555.
  TRAPPE J. (2009). The hunted: commercially attractive truffles native to North America. In: Les
    champignons forestiers comestibles à potentiel commercial. Biopterre, ACCHF, Université Laval, CEF,
    RNC. pp. 1-5.
  WEDEN C., PETTERSSON L., DANELL E. (2009). Truffle cultivation in Sweden: results from Quercus robur
    and Corylus avellana field trials on the island of Gotland. Scan J For Res 24: 37–35.
  ZAMBONELLI A., IOTTI M., PIATTONI F. (2012). Chinese Tuber aestivum sensu lato in Europe. Open Mycol J
    6: 22-26.
  ZAMBONELLI A., IOTTI M., ROSSI I., HALL I. (2000). Interaction between Tuber borchii and other
    ectomycorrhizal fungi in a field plantation. Mycol Res 104(6): 698-702.
  ZAMBONELLI A., IOTTI M., GIOMARO G., HALL I., STOCCHI V. (2002). T. borchii cultivation: an interesting
    perspective In: Edible mycorrhizal mushrooms Proceedings of 2nd international workshop on edible
    ectomycorrhizal mushrooms (Hall I, Wang Yun, Danell E, Zambonelli A eds). New Zealand Institute for
    Crop and Food Research, Christchurch New Zealand. CD ROM.
  ZHANG J.P., LIU P.G., CHEN J. (2012). Tuber sinoaestivum sp. nov., an edible truffle from southwestern
    China. Mycotaxon 122: 73-82.




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