Keywords

1 Introduction

A hypogenous life cycle is common in nearly every major group of fleshy fungi within the phyla Ascomycota , Basidiomycota and Mucoromycotina (Tedersoo et al. 2010). Underground fruiting structures are formed in most of the Ascomycetes families (Trappe et al. 2009) but true truffles are considered to be only those species in the genus Tuber, family Tuberaceae, order Pezizales (Zambonelli et al. 2016). The origin of the genus Tuber is estimated to be in the early Cretaceous, around 142 Mya (Bonito et al. 2010; Jeandroz et al. 2008) evolving from an epigeous ancestor (Bonito et al. 2010). Spores are sequestered inside the fruiting bodies and generally are dispersed by mycophagous animals that are attracted by a wide range of powerful aromas (Zambonelli et al. 2017). Most truffles live in ectomycorrhizal (ECM) relationships with shrubs and trees in temperate forests mostly in the Northern Hemisphere although the desert truffles inhabit semi deserts of the northern Mediterranean and Africa (Bonito et al. 2013).

The genus Tuber includes around 200 species (Bonito et al. 2010) but only a handful have economic value, with the European species the most valuable, such as the white truffles T. magnatum Picco (Italian white truffle) and T. borchii Vittad. (bianchetto truffle ) and the black truffles T. melanosporum Vittad. (Perigord black truffle) and T. aestivum Vittad. (summer truffle ). Other edible black truffles that are locally commercialized in Europe are T. brumale Vittad. (winter truffle ), T. mesentericum Vittad. (truffe mèsentèrique) and T. macrosporum Vittad. (smooth black truffle) (Hall et al. 2007). Several other Tuber species from China, such us the Chinese black truffles, T. indicum Cooke and Massee, T. sinoaestivum J.P. Zhang and P.G. Liu, the Chinese white truffle T. panzhihuanense X.J. Deng and Y. Wang (Deng et al. 2013; Wang 2012; Zhang et al. 2012) and white North American truffles such as T. oregonense Trappe, Bonito and P. Rawl., T. gibbosum Harkn. and T. lyonii Butters (Lefevre 2012) are locally consumed or exported to Europe where they are often passed-off as their more expensive European cousins.

In this chapter, the new biotechnological approaches for improving cultivation techniques and for germplasm conservation of the European white truffles Tuber magnatum and T. borchii are detailed and their very different lifestyles contrasted.

2 Tuber magnatum and T. borchii Characteristics

2.1 Morphology

Truffles present different morphological features during the three different phases of their life cycles: vegetative as free-living mycelium, symbiotic as ectomycorrhizas, and reproductive ascomata (the fruiting bodies).

The hyphae of all Tuber spp. are 3–8 μm in diameter, septate, hyaline and simply branched, showing frequent anastomoses, rare coils and vesicles (Fig. 14.1a) (Iotti et al. 2002). In T. borchii vesicles are more numerous, assuming the characteristics of chlamydospores in unfavorable conditions such as in the presence of adverse bacteria (Barbieri et al. 2005), when cultures age (Iotti et al. 2002) or when iron is deficient (Picceri et al. 2018). The mycelial colonies are regular, whitish in color with most of the biomass growing into the agar or completely submerged when in liquid culture. In contrast, the fruiting bodies and the mycorrhizas differ greatly and are characteristic for each species.

Fig. 14.1
figure 1

Morphological features of the Tuber magnatum and T. borchii : (a) T. borchii mycelium, (b) T. magnatum ascoma, (c) T. magnatum spores, (d) T. magnatum mycorrhizas, (e) T. borchii ascomata, (f) T. borchii spore, (g) T. borchii mycorrhiza . (Figure constructed by A. Zambonelli)

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Despite the often very dark to black patches on the surfaces of Tuber borchii and T. magnatum both are regarded as white truffles because both have ascomata with a smooth peridium and are generally straw-colored. In contrast, the black truffles have a brown to black peridium ornamented by warts, for example, T. melanosporum, T. aestivum, T. mesentericum and T. brumale. The peridium of T. magnatum is smooth, olive-yellow in color; the gleba at maturity is ocher to pale brown marbled with numerous white thin sterile veins (Fig. 14.1b). Occasionally, T. magnatum ascomata show pink to red patches on the surface and within the gleba, probably due to bacterial infection by Microbacterium and/or Chryseobacterium (Amicucci et al. 2018). However, these do not appear to affect the aromatic characteristics of the ascomata. The spores are subglobose to broadly ellipsoid, light-yellow, yellow-ocher at maturity, ornamented with an irregular reticulum (2–) 3–5 (−7) μm high, with 2–4 meshes across the width of the spores. (Fig. 14.1c). In contrast, the peridium of T. borchii is minutely pubescent caused by a layer of yellow reddish-brown colored hyphae with setose hairs and the gleba at maturity is dark brown with wide, white veins (Fig. 14.1e). The spores are ellipsoid to broadly ellipsoid, ocher-brown at maturity, ornamented with a regular reticulum with meshes 3–7 (−10) μm high, 3–10 across the width of spores (Fig. 14.1f) (Zambonelli et al. 2000a).

The mycorrhizas of both species are macroscopically similar, simple or ramified with whitish-ocher, fulvous colors, in Tuber borchii , and ocher to grayish-white in T. magnatum (Mello et al. 2001; Zambonelli et al. 1993). Microscopically the mantles are similar, composed of epidermoid cells ornamented by awl-shaped hyaline cystidia. These are never ramified in Tuber borchii ectomycorrhizas and 70–140 μm long (Fig. 14.1g), whereas they are often branched in T. magnatum and up 480 μm long (exceptionally 580 μm) (Giomaro et al. 2000; Mello et al. 2001; Riccioni et al. 2016) (Fig. 14.1d).

2.2 Distribution

Until the turn of the millennium Tuber magnatum was considered an almost exclusive Italian truffle restricted to the north and center of the country with smaller patches, in Istria, Croatia southeastern France and the Ticino Canton of Switzerland (Hall et al. 1998). However, recent studies have found it in Sicily (Vasquez et al. 2014), the Geneva Canton of Switzerland (Büntgen et al. 2019), Hungary and several Balkan regions (Bratek et al. 2007; Riccioni et al. 2016). Intriguingly, is has also been found in Thailand (Suwannarach et al. 2017). In contrast, T. borchii has one of the widest distributions of any truffle being found throughout Europe, from southern Finland to Sicily and from Ireland to Hungary and Poland (Hall et al. 2007; Shamekh et al. 2009) (Fig. 14.2).

Fig. 14.2
figure 2

Geographical distribution of Tuber magnatum (green symbols) and T. borchii (brown symbols). (Sources: Gogan-Csorbai et al. 2018; Hall et al. 2007)

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2.3 Economic Importance

The high economic value of truffles is due to their unique aromas and flavors, which make them one of the most expensive food delicacies. Tuber magnatum is the truffle that commands the highest prices although this varies depending on the size of the ascoma and from season to season. In poor harvest years such as 2017, the retail prices in Italy were very high ranging between 3500 EUR/kg and 5000 EUR/kg. The production in 2018 was greater and prices fell to 1000–2100 EUR/kg (https://www.termometropolitico.it/1332538_2019-05-02-prezzo-tartufo-bianco.html). Outside Italy even higher prices can be paid, for example, T. magnatum was sold in Harrods, London, for GBP 6500/kg on 25 September 2010 (Badalyan and Zambonelli 2019; Hall, personal communication). Tuber borchii commands lower prices than T. magnatum and T. melanosporum, although recently it has become more appreciated and prices have risen somewhat. In 2019 retail prices in Italy were 105–305 EUR/kg depending on the size (https://www.andareatartufi.com/wordpress/quotazioni-tartufo-bianchetto/rom). In contrast, the off-season price of T. borchii produced in New Zealand that are not mixed with inferior species of white truffle can fetch much higher prices that hover around NZD 3000/kg (about 1700 EUR/kg (http://www.trufflesandmushrooms.co.nz/Tuber%20borchii%20web.pdf). Unlike black truffles, white truffles are used either uncooked or added to dishes just after cooking in order to retain the delicate, volatile aromas such as when flavoring pasta or salads.

2.4 Ecology

Tuber magnatum has specific ecological requirements, which limit its distribution in Europe. It is generally found below 600 m elevation, but in Southern and Central Italy it can be found up to 900 m elevation. It develops in woods with more or less closed canopies, as well as areas with relatively sparse vegetation. It often fruits in floodplains or around stream beds probably because its mycelium displays a high demand for water for growth in soil (Iotti et al. 2018; Marjanović et al. 2015). The host plants are: Alnus cordata Desf. (Italian alder), Corylus avellana L. (hazelnut), Ostrya carpinifolia Scop., Populus alba L. (white poplar), P. tremula L. (European trembling aspen), P. nigra L. (Lombardy poplar), Quercus cerris L. (Turkish oak), Q. ilex L. (holm oak), Q. pubescens Willd. (downy oak), Q. robur L. (English or common oak), Salix alba L. (white willow), S. caprea L. (pussy willow), Tilia cordata Mill. (small-leaved lime), T. platyphyllos Scop. (large-leaved lime) (Hall et al. 2007) and Fagus sylvatica L. (Büntgen et al. 2019). It is also, though rarely, found under Pinus spp. (Zambonelli, personal communication) but the ectomycorrhizal association with conifers has never been verified with molecular tools. Tuber magnatum requires well-drained soils without gravels, having a preference for neutral to extremely alkaline soils (pH 7–8.5), with abundant active CaCO3 (Hall et al. 1998). The peculiar characteristics of T. magnatum soils are good aeration and very soft consistency due to its elevated volume (around 15%) occupied by interconnected macropores (Bragato and Marjanović 2016). The climatic requirements include a winter temperature range of 2–8 °C (in January) and summer temperatures of 18–26 °C (in June). Ideal precipitation ranges between 500–2000 mm and spread more or less evenly throughout the year, although in summer this tends to be in the form of thunderstorms (Hall et al. 2007). The ascomata are sensitive to winter frost and summer drought which helps explain their preponderance in riparian habitats (Iotti et al. 2018; Le Tacon 2016).

Tuber borchii has broad ecological requirements and is found from sea level to 1100 m elevation (Tanfulli et al. 1999). It forms ectomycorrhizal associations with a wide range of host plants including several species of conifers including pines and cedars (Table 14.1). It is most commonly found on Pinus spp. particularly P. pinea and P. pinaster in the coastal areas of the Mediterranean Sea. Recently it was found to form mycorrhizas with commercially interesting fruit plants such as the strawberry tree (Arbutus unedo, Lancellotti et al. 2014) and pecan (Carya illinoinensis, Benucci et al. 2012). Surprisingly, it can also form mycorrhizas with orchids (Tešitelová et al. 2012) confirming its capacity to form symbiotic associations with herbaceous plants, as predicted by Mannozzi-Torini (1988).

Table 14.1 Putative host plants of the white truffles
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Tuber borchii grows in moderate to strongly calcareous, very sandy to silty soils (Fig. 14.3) such as those in coastal areas of Italy, as well as in the same alkaline soils where the Italian white truffles are harvested in the Apennines. In Italy it is also found in neutral and slightly acidic soils where the pH ranges from 6–7. Occasionally the pH can be as low as 5.2 (Gardin 2005) but such soils are more likely to be the home of inferior white truffles such as Tuber maculatum Vittad. (Hall, personal communication). The climate of areas where bianchetto is found in Italy and New Zealand ranges from cool temperate to Mediterranean, with annual rainfall of 600–1600 mm.

Fig. 14.3
figure 3

Soil textural triangle for the bianchetto truffle . The area marked in violet is suitable for this truffle . The arrows indicate the direction the reader needs to read the graph. (Source: Hall et al. 2007)

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2.5 Aroma Characteristics

The aroma of truffles is due to the volatile organic compounds (VOCs) which are produced during ascoma formation and maturation. Aroma production is a biological strategy used by truffles to attract mycophagous animals which disseminate their spores (Zambonelli et al. 2017). Although around 300 VOCs have been reported in truffles (Splivallo et al. 2011) only 10–20 odorants per species are responsible for the typical truffle odors perceived by the human nose (Schmidberger and Schieberle 2017). Some of these are common to several truffle species particularly 2- and 3-methylbutanal, 2- and 3-methylbutan-1-ol and oct-1-en-3-ol. Others are species-specific such as 2,4-dithiapentane in Tuber magnatum and thiophene derivatives in T. borchii (Fiecchi et al. 1967; Gioacchini et al. 2005; Splivallo and Ebeler 2015; Splivallo et al. 2011).

Truffle products aromatized using synthetic scents are quite different and generally less appealing than fresh ascomata, primarily because they contain a markedly higher quantity of 2,4-dithiapentane and two VOCs , dimethyl sulfoxide and dimethyl sulfone, which are absent from ascomata (Wernig et al. 2018).

Not only does genotype affect aroma (Splivallo et al. 2012) but also associated bacteria, yeasts and other fungi that also produce VOCs and contribute to truffle aromas (Buzzini et al. 2005; Pacioni and Leonardi 2016; Splivallo and Ebeler 2015; Splivallo et al. 2014; Vahdatzadeh et al. 2015). Truffle aroma is also influenced by other environmental factors such as soil composition, climate and the host plant (Splivallo and Culleré 2016). It is therefore not surprising that as ascoma maturity, methods for storing, changes in microbiome composition, season and geographical location, all affect VOCs profiles and perceived quality (Gioacchini et al. 2008; Pennazza et al. 2013; Vahdatzadeh et al. 2019; Vita et al. 2015; Zeppa et al. 2004). Mycelial fermentation techniques have been used to produce natural truffle aromas but these were found to be less complex (Splivallo et al. 2007; Tirillini et al. 2000). For example, 3-methyl-4,5-(2 H)thiophene is produced only during the sexual stage of Tuber borchii fruiting body formation. However, the VOCs composition of Tuber mycelium can be improved by supplying axenic cultures of truffle mycelium with leucine, isoleucine, phenylalanine and methionine inducing the synthesis of the VOCs derived from amino acid catabolism (Ehrlich pathway) (Li et al. 2012; Liu et al. 2013; Splivallo and Maier 2016; Vahdatzadeh and Splivallo 2018; Xiao et al. 2015). It is hoped that strain selection may be used to improve truffle mycelium VOC production (Vahdatzadeh and Splivallo 2018) as well as repeated freeze-thaw cycles (Xiao et al. 2015).

This capacity of Tuber mycelium to produce VOCs may be commercially exploitable and lead to the production of natural flavor with a higher consumer acceptance of truffle-flavored food products (i.e. truffle-flavored olive oil) that currently predominantly contain synthetic flavors (Splivallo and Maier 2016).

3 Cultivation

3.1 Traditional Cultivation Practices

The first techniques for cultivating truffles were developed for Tuber melanosporum, possibly independently by Josef Talon and Pierre Mauléon in the nineteenth century (Hall and Zambonelli 2012). They simply sowed acorns collected under oaks that were producing truffles and then transplanted these into adjacent soils. Only in the 1970s was modern truffle cultivation introduced into France and Italy after the discovery of the mycorrhizal nature of truffles. This consists of producing mycorrhizal plants in the greenhouse and then out planting them into areas with suitable soils and climate. Using these techniques, T. aestivum, T. borchii , and T. brumale are now cultivated as well as T. melanosporum, although the latter is still the most extensively cultivated, not only in Europe but in countries outside of Europe (Hall et al. 2017; Reyna and Garcia-Barreda 2014). In recent years T. borchii cultivation has become more widespread in Italy (Zambonelli et al. 2015), Spain, Portugal (https://micofora.com/en/growing-tuber-borchii/) and in France. Tuber borchii is also successfully cultivated in New Zealand, Australia the USA (Zambonelli et al. 2015) and China (Wang Yun, personal communication).

The production of Tuber mycorrhizal plants represents the first important step in modern truffle cultivation. The plants are preferably colonized with a single Tuber species of interest and are preferably free of other contaminating ectomycorrhizal fungi. Generally, the quantification of mycorrhizal formation is assessed morphologically. Under a stereomicroscope the percentage of root tips colonized by Tuber spp. is counted or estimated (Andrés-Alpuente et al. 2014) although the use of this method requires trained personnel (Sisti et al. 2010).

Although, the link between the level of mycorrhizal formation on inoculated seedlings prior to planting has not been conclusively demonstrated, planting seedlings with high degrees of truffle mycorrhizal colonization is recommended to improve the chance of truffle production in plantations (Murat 2015). For this reason in Italy and France Tuber mycorrhizal plants can be commercialized only if the degree of mycorrhization (number of root tips colonized with Tuber/total number of tips ×100) is above 30% (Donnini et al. 2014; Govi et al. 1997; Regione Emilia-Romagna 2018). Tuber borchii commercialized plants usually exceed this level of mycorrhizal infection. In contrast, the mycorrhizas of T. magnatum can be difficult to obtain and only a company in France is actually selling T. magnatum mycorrhizal plants at prices 5–10 times higher than T. borchii or black truffle mycorrhizal plants. Morphological and molecular methods have now been perfected in order to identify and quantify mycorrhizal formation on root systems (Alvarado and Manjon 2013; Andrés-Alpuente et al. 2014; Fischer and Colinas 1996; Mello et al. 2006; Rocchi et al. 1999; Zambonelli et al. 1993, 2012a). In contrast, to ectomycorrhizas of black truffles (T. aestivum, T. brumale and T. melanosporum) (Zambonelli et al. 1993), the morphological characters of all white truffles are similar, and in particular the mycorrhizas of T. maculatum, T. oligospermum, T. dryophilum and T. borchii (Boutahir et al. 2013; Zambonelli et al. 1999) which require the use of molecular techniques to be sure. Species specific primers for T. borchii and T. magnatum were designed to be used in simple, multiplex or quantitative PCR (Amicucci et al. 1998, 2000; Iotti et al. 2012a; Mello et al. 1999; Rubini et al. 2001) (Table 14.2). Spore inoculation is used by the vast majority of companies that produce Tuber mycorrhizal plants because it is simple and effective. First, fresh, dry or frozen truffles are ground in water to create a spore suspension which is then used to inoculate sterile seedling or cuttings a few months old. However, with spore inoculation techniques, plants can become contaminated with undesirable truffle species if the truffles are not carefully selected and identified. Moreover, pests and pathogens can be introduced with the inoculum. Furthermore, because ascospores are produced sexually, each plant is potentially colonized by a different suite of fungal genotypes from every other with unknown characteristics.

Table 14.2 Molecular markers used for genetic characterization in Tuber borchii and T. magnatum
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In order to achieve ascoma production the infected plants have to be planted in the field in environmental conditions (soil, climate) that suit both the host plant and the truffle . Tuber borchii tends to fruit more quickly than T. melanosporum. In experimental Pinus pinea plantations the first ascomata were found only 3–4 years after planting (Hall, personal communication; Zambonelli et al. 2000b). Although, T. borchii cultivation is becoming more common, no specific research on how to manage T. borchii cultivation has been carried out and no handbook on T. borchii cultivation is available in the market, although a booklet is available from Ian Hall (2017) that reviews what are thought to be the ideal soil and climatic conditions. However, farmers tend to copy guidelines for T. melanosporum cultivation which in low pH soils can result in excessive applications of lime and depressed soil trace element concentrations.

Some success has recently been reported by Bach et al. (2021) in the production of T. magnatum  in France and it is to be hoped that this will lead to its routine cultivation rather than the erratic positive results obtained in Italian experiments of the 1990s and 2000s (Bencivenga et al. 2009).

3.2 New Biotechnologies for Cultivating Tuber borchii

Tuber borchii is considered the model species for genetic and biological studies on truffles because its mycelium can be cultured in vitro more easily than other species of truffle . In contrast, difficulties in making pure cultures of T. magnatum have always hindered insights into its biology.

The first mycorrhizal plants with Tuber borchii mycelium were produced in greenhouses on Pinus strobus and poplar by the Italian researchers Fontana and Palenzona (1969). Later a technique to produce Tuber mycorrhizal plants under in vitro conditions on Populus alba micropropagated plantlets was perfected by Zambonelli et al. (1989), and later on with Tilia platyphyllos (Sisti et al. 1998). The method involves raising micropropagated plantlets in a medium with a low concentration of auxin (0.2 mg/l of NAA) to allow the differentiation of roots with multiple secondary roots with limited growth. The mycelium of T. borchii is then isolated onto PDA agar in Petri dishes and then bulked up in MMN liquid medium. The mycorrhizal synthesis is performed in vermiculite moistened with MS/2 liquid medium at pH 6.3 (Giomaro et al. 2005). Subsequently, Tilia platyphyllos-Tuber borchii mycorrhizal plants became a model to study plant/fungus interactions (Menotta et al. 2004; Polidori et al. 2002, 2007; Zeppa et al. 2002).

Tuber borchii mycorrhizal plants inoculated with mycelia have never been produced commercially for a variety of reasons. Spore inoculation is easier and gives good results with T. borchii . Moreover, nurseries producing Tuber mycorrhizal plants are usually not equipped with laboratories for isolating and cultivating Tuber mycelium in vitro. Although T. borchii mycelium generally grows more quickly than other Tuber spp. (Iotti et al. 2002) it is still more difficult to manipulate compared with other mycorrhizal mushrooms. It was not until a few years ago that there was evidence that plants inoculated with mycelial-pure cultures could produce truffles. In fact, Paolocci et al. (2006) hypothesized, and later confirmed by Martin et al. (2010), that when the T. melanosporum genome was sequenced and the mating type idiomorphs characterized, that truffles were heterothallic and that two strains carrying different mating types would be needed to mate in order to produce the fruiting bodies (de la Varga et al. 2017; Rubini et al. 2014).

Iotti et al. (2016) reported that the first experimental Tuber borchii truffle orchard inoculated with 5 different T. borchii strains individually or mixed had started to produce and 99 ascomata were eventually collected between February and April with a total fresh weight of 722 g. This demonstrated for the first time that it was possible to produce truffles with mycorrhizal plants inoculated with pure cultures. Subsequently, a study conducted on this truffière by Leonardi et al. (2019) showed that the paternal genotypes originated by recombination of the inoculated strains and only in a few cases from alien genotypes. Furthermore, 4 of the 5 inoculated strains persisted 9 years after establishing the plantation and remained confined to their inoculated plants without migrating into the neighboring strain areas.

The isolation and genetic characterization of different strains from geographical regions with varying soil and climatic conditions could lead to strains that produce ascomata with enhanced organoleptic characteristics and suited to particular environmental conditions. Recent work conducted by Vahdatzadeh and Splivallo (2018) on the mycelium of nine Tuber borchii strains has highlighted how genetically-different strains produce different aromas. Other work conducted by Leonardi et al. (2017) has shown how T. borchii strains differ in their tolerance to heat stress, demonstrating that it should be possible, in this regard at least, to select the most suitable strains for a specific cultivation area.

4 Genetic Diversity and Germplasm Conservation

4.1 Genetic Diversity

A proper understanding of genetic variation within and among Tuber species and populations, as well as their phylogeography, is indispensable for efficient utilization of a valuable truffle .

The first studies on the genetic diversity of Tuber species involved the analysis of random amplified polymorphic DNA (RAPD ) and sequence characterized amplified region (SCAR) markers (Amicucci et al. 1997; Bertini et al. 1998; Gandeboeuf et al. 1997a, b; Lanfranco et al. 1993; Potenza et al. 1994; Rossi et al. 2000). However, ITS regions of nuclear rDNA, alone or in combination with other loci, quickly become the elective markers to explore the genetic diversity within Tuber species and to infer their phylogenetic relationships (Amicucci et al. 1996; Bonito et al. 2010; Bonuso et al. 2010; Guillemaud et al. 1996; Halász et al. 2005; Henrion et al. 1994; Huang et al. 2009; Iotti et al. 2007; Mello et al. 1996; Merényi et al. 2014; Murat et al. 2004, 2005; Paolocci et al. 1995, 2004; Qiao et al. 2018; Riccioni et al. 2019; Sica et al. 2007; Wang et al. 2006; Wedén et al. 2005). Microsatellite markers were first investigated for truffle typing (Amicucci et al. 2002; Longato and Bonfante 1997) but they were found more useful for studying population genetics (Bertault et al. 2001; Figliuolo et al. 2013; Leonardi et al. 2019; Linde and Selmes 2012; Molinier et al. 2015, 2016a, b; Murat et al. 2013; Riccioni et al. 2008; Rubini et al. 2005; Taschen et al. 2016).

The first studies of Tuber magnatum and T. borchii have surprisingly deduced that the two species fall into two distinct clades of the genus Tuber (Percudani et al. 1999). Tuber magnatum is co-located inside the Aestivum clade which includes species that are morphologically quite different: T. aestivum, T. mesentericum and T. sinoaestivum, three species of black truffle characterized by a warty peridium and reticulate ascospores, and T. panniferum which is a unique truffle with a wooly peridium and very spiny ascospores (Bonito and Smith 2016; Bonito et al. 2013; Jeandroz et al. 2008). In contrast, T. borchii is co-located inside the Puberulum clade, which includes at least 63 morphologically-similar species distributed across Europe, Asia, North America, South America and North Africa (Bonito et al. 2013; Lancellotti et al. 2016).

Several studies on Tuber magnatum genetic intraspecific variability have been carried out to find molecular markers that may define its geographic origin. The first studies, carried out by RAPD (Amicucci et al. 1997; Gandeboeuf et al. 1997a; Lanfranco et al. 1993; Pomarico et al. 2007; Potenza et al. 1994) showed a very low intraspecific polymorphism in T. magnatum with respect to other Tuber spp. This low genetic diversity in T. magnatum was later confirmed by several researchers (Iotti et al. 2012a; Mello et al. 2005; Pomarico et al. 2007) who analyzed ITS regions and other genetic markers. Only simple sequence repeat loci (SSR ) proved to be suitable as markers for population genetic studies of T. magnatum . Rubini et al. (2004) first selected 8 polymorphic SSRs and used them to analyze 370 ascomata from 5 main areas. They defined 28 populations, one of which was from the Istria region of Croatia and 4, 12, 10 and 1 from the north, center, center-south and south of Italy, respectively.

Recently a new transcriptome assembly of the Italian white truffle has allowed the identification of 2581 gene-based SSR markers (Vita et al. 2018). In addition, the T. magnatum genome has now been sequenced (Murat et al. 2018a) and a bioinformatics workflow was applied in order to mine for microsatellite sequences. As a result, 11,189 microsatellite markers specific to T. magnatum were found with 3377 marker loci matching with T. magnatum proteins (Uncu and Uncu 2019). This will provide some important new tools to explore T. magnatum genetic diversity and to find genetic markers that might define the genetic origin of the ascoma.

After the first studies on the genetic diversity of Tuber borchii using RAPD (Bertini et al. 1998; Gandeboeuf et al. 1997a) and IGS (Ciarmela et al. 2002), a multigene phylogeny was carried out on 61 representative specimens with a broad distribution throughout Italy (Bonuso et al. 2010). In this study two cryptic species of T. borchii were identified.

Recently the Tuber borchii genome has also been sequenced (Murat et al. 2018b) and showed 1111 SSRs. Among them 14 SSRs were highly polymorphic and were able to differentiate single genotypes after testing on 50 ascomata harvested in Italy and Hungary (Leonardi et al. 2019).

4.2 Genetic Resources Conservation Approaches

The conservation of truffle genetic resources is becoming more important than ever because of the increasing threat from deforestation, overharvesting, climate warming and changing agricultural practices.

Truffle cultivation could be an efficient method to ex situ conserve Tuber genetic resources (Varese et al. 2011). To achieve this goal, it would be important to use only spore inoculation methods from locally-harvested truffles in order to secure their genetic diversity. Although truffles growing in natural areas are assumed to be genetically adapted to the site’s climatic and edaphic conditions, it is also possible that they represent the vestiges of a distribution when conditions may have been quite different. Regardless, the creation of a germplasm bank of Tuber spp. is an imperative in the future once mycelial inoculation becomes the method of choice for nurseries producing Tuber mycorrhizal plants for cultivation.

4.2.1 In Vitro Conservation of Tuber spp. Genetic Resources

In the conservation of mycelial pure cultures in vitro it is very important to ensure that their purity, vitality and genetic integrity is maintained over time (Smith 2004; Voyron et al. 2007). Currently, many conservation methods are available (Nakasone et al. 2004) and the choice of the most appropriate method should depend on the species under consideration and on the purpose of conservation.

The short-term conservation of Tuber mycelial subcultures, obtained by taking small plugs of mycelium from the edge of a colony, is easiest and inexpensive, but the risk of nutrient medium contamination is high. Furthermore, Tuber mycelium is known to grow very slowly in pure culture (Iotti et al. 2002, 2012b) and repeated transfers may lead to the loss of infectivity, viability and induce mutation or changes in gene expression (Coughlan and Piché 2005; Smith 2012).

Another preservation technique is cryopreservation in liquid nitrogen, which consists of preserving the biological material at ultra-low temperature after the addition of several cryoprotectants. In this process, the material is gradually cooled down to a temperature close to −196 °C, and then stored at that temperature. In this condition all biological activity, including biochemical reactions that would lead to cell death, are blocked and prevents any cellular and genetic damage (Smith et al. 2001). Moreover, cryopreservation reduces work load and saves space needed for preservation on agar, ensures the continued infectivity of strains, and streamlines the accessibility of the collection. The greatest disadvantages of this technique are the large initial investment and ongoing operating costs primarily for the continuous supply of liquid nitrogen which needs to be constantly controlled and maintained. Cryopreservation of Tuber borchii mycelium was tested for the first time by Stielow et al. (2012) although the survival ratio on charcoal filter paper strips after thawing was low. In 2017, Piattoni et al. (2017) used a different cryopreservation protocol, employing sorbitol, sucrose and dimethyl sulfoxide as cryoprotectans which allowed cryopreservation of T. borchii mycelium without reducing its infectivity.

A similar technique to that of cryopreservation, with the high survival rate of biological material, is ultra-freezing (Kitamoto et al. 2002). This differs from cryopreservation at a higher storage temperature, ranging from −80 to −130 °C and the use of an ultra-freezer instead of liquid nitrogen. This technique, applied to various ectomycorrhizal mushrooms, has shown differing results depending on the cryoprotectans used and the protocol applied (Crahay et al. 2013; Kitamoto et al. 2002; Obase et al. 2011). A recent protocol, applied to the medicinal mushroom Ganoderma lucidum (Curtis) P. Karst., developed by Leonardi et al. (2018) also showed very promising results with Tuber spp. and currently tests are underway to verify the survival of Tuber spp. mycelia at ultra-freezing temperatures.

4.2.2 In Situ Conservation of Tuber magnatum

Tuber magnatum has yet to be successfully cultivated so the only way to commercially exploit it without damaging natural production is to conserve it in situ. Intensive harvesting of truffles has been shown to cause decreases in truffle production in Europe and China as a consequence of physical damage to Tuber ectomycorrhizas, the soil mycelium and habitat, and perhaps suppressing fertilization. In order to protect this important resource, it is essential to enforce strict rules during harvest to protect their unique environment, to ban the harvesting of unripe ascomata by, for example, raking and to stop overharvesting in some areas allowing spore diffusion.

Regretably, fungi seldom receive legal protection and examples of in situ conservation are rare (Venturella et al. 2011) despite the Italian national law (L. 752/1985, https://www.gazzettaufficiale.it/eli/id/1985/12/21/085U0752/sg) that covers the most important rules for harvesting, cultivation, conservation and marketing of truffles. Some positive aspects of this law are, for example, that immature truffles are protected, dogs (rather than pigs) must be used to locate truffles, a special small trowel must be used to excavate the truffles and excavations are to be covered immediately after a truffle is harvested to avoid damage to the mycorrhizas. However, the biggest concern for T. magnatum in situ conservation is the ever-increasing number of truffle harvesters, which currently stands at around 45,000 in Italy (Ministry of Agriculture, Food and Forestry Policies and Tourisms https://www.politicheagricole.it/flex/cm/pages/ServeBLOB.php/L/IT/IDPagina/11100). Sadly, these collectors have open access to extensive oak forests and woodlands and other lands not under cultivation and occupied by T. magnatum . Furthermore, there is a lack of political motivation to reduce the number of collectors or to create truffle reserves or ecological corridors, where truffle harvesting is forbidden.

The preservation and plantation of Tuber magnatum host plants and the protection of the specific truffle environments are the only effective conservation measures proposed in some parts of Italy. In some municipalities of Tuscany, for example, the owners of T. magnatum productive trees receive a reward if they do not cut the trees. In some regions, the local government can give landowners the exclusive right to collect truffles but only after it has been verified that their productive forest has been significantly improved through an adequate thinning of the undergrowth.

Until recently, traditional cultural practices have often been applied in an attempt to improve Tuber magnatum areas without knowledge of their subsequent effects. This is because T. magnatum ectomycorrhizas are rarely detected in the field (Leonardi et al. 2013; Murat et al. 2005) and so cannot be used as an indicator of its diffusion in soil, and the production of ascomata is too scattered and variable to follow in short-term experiments.

However, a molecular monitoring method based on quantitative PCR has been perfected (Iotti et al. 2012a) in a recent Italian regional project (MAGNATUM) that allows the quantification of the mycelium of Tuber magnatum in soil. For the first time we can now develop a simulation model to predict the mycelial dynamics of T. magnatum at varying soil temperatures and moisture conditions (Iotti et al. 2018) and measure the effects of managing summer irrigation. This also provides the first management guidelines aimed at maintaining and improving the environmental conditions we believe are suited to T. magnatum , such as soil tillage to relieve soil compaction (Salerni et al. 2014) and the selective removal or thinning of invading shrubs like brambles (Rubus spp.) (Zambonelli et al. 2012b).

5 Conclusion and Prospects

Tuber magnatum and T. borchii are two morphologically-similar white truffles which are traditionally important natural economic resources in Italy and a few other European countries. However, the less valuable T. borchii has become increasingly popular over the past 20 years following its successful cultivation first in Italy, followed by New Zealand. It has a very wide ecological range and can be easy cultivated. In contrast, T. magnatum has defied routine cultivation but there are few signs that it will be commercially cultivated anytime soon.

In addition to traditional cultivation methods, new techniques are also available for Tuber borchii . In particular, a technique for inoculation of plants with pure cultures has been perfected that opens up the possibility of selecting genotypes with particular characteristics, for example, specific climatic or edaphic zones.

The genome sequencing of Tuber borchii should help reveal the set of genes that control the adaptability of T. borchii to different environmental conditions and its productivity and quality . Genetic transformation of Tuber mycelia has been scarcely investigated (Grimaldi et al. 2005; Poma et al. 2005) and the proposed techniques not further developed. However, the discovery of the CRISPR/Cas9 immune system of bacteria and archaea and their repurposing for genome editing has elicited a new era in genetic engineering for filamentous fungi (Kwon et al. 2019) that may be applied to improve the characteristics of isolated Tuber strains increasing their ability to produce aromas and/or their adaptability to extreme climatic conditions. This might increase the cultivation area and help to overcome the risks of declining truffle production in the southern parts of Europe caused by predicted global warming (Thomas and Büntgen 2017). Obviously, to better exploit its genetic variability, the creation of a germplasm bank to preserve T. borchii genetic resources is mandatory. Cryopreservation techniques of mycelia have been shown to be an efficient tool for long-term preservation of truffle strains without affecting their viability and infectivity.

The cultivation of Tuber magnatum may receive a boost in the new genomic era. For example, comparative genomics could help to identify those genes which control T. magnatum mycorrhiza formation and nutrition, and to find conditions that favor its mycorrhiza development and in vitro cultivation of mycelia.