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2 Tuber melanosporum: The Black Perigord Truffle

2 Tuber melanosporum: The Black Perigord Truffle

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22



J. Chen et al.

Tuber_hiemalbum_FM205571



Tuber_hiemalbum_FM205570



64 Tuber_melanosporum_KF591751



Tuber_melanosporum_KM659874



100

Tuber_melanosporum_AJ583649



95



66



Tuber_melanosporum_KM659870



Tuber_melanosporum_JF264440



100



Tuber_indicum_JQ638998



Tuber_regimontanum_NR_121340



100

75



Tuber_brumale_KM659875



Tuber_brumale_KF551019



Tuber_pseudoexcavatum_DQ329374



Tuber_magnatum_JX087962



Fig. 2.1 Neighbour-joining phylogenetic tree realised with the ITS regions of the rDNA. The

phylogeny was realised with MEGA6. The bootstrap value above 60 is indicated:



2.2.2



Tuber melanosporum Production in Truffle Orchards



Currently, T. melanosporum production occurs primarily in truffle orchards. The

cultivation of this species is based on the planting of seedlings that have been

previously inoculated with truffles in controlled conditions, such as nurseries. The

inoculation of seedlings with T. melanosporum is performed using ascomata as

inoculum. Ascomata contain meiotic spores, which, after germination, form ECMs

on seedling roots (Paolocci et al. 2006). Seedlings are then grown in greenhouses

and sold following a procedure of quality control (Murat 2015). This inoculation

technique, developed in Italy and France, is now used worldwide (Chevalier and

Grente 1979). In Europe it is estimated that more than 500,000 seedlings inoculated

with truffles are sold each year; this compares to the roughly 100,000 inoculated

seedlings sold annually on other continents (Murat 2015).

In the twentieth century, the vast majority of truffle production moved from

secondary woodlands to planted truffle orchards. Aside from truffle production,

truffle orchards help transform agricultural landscapes into sustainable and productive agroforestry ecosystems with high value added; this transition promotes landscape management in fire-prone regions and increases biodiversity compared to that

of intensive agriculture (Therville et al. 2013). As a form of agroforestry, truffle



2 The Black Truffles Tuber melanosporum and Tuber indicum



23



orchards may also help mitigate climate change through carbon sequestration

(Hamon et al. 2009) and other ecosystem services such as improvements in water

and soil quality (Alam et al. 2014). All these aspects should be highlighted together,

with truffle production, to convince farmers, foresters and local agencies to invest in

T. melanosporum cultivation.

Tuber melanosporum production in truffle orchards faces two important bottlenecks: the initiation of sexual reproduction and the growth of ascomata during a

period of several months linked to a tree (Fig. 2.2). To complete their life cycle,

truffles must associate with a mature tree. It has been shown that the carbon

supporting T. melanosporum ascomata comes exclusively from tree photosynthesis

throughout a continuous association (Le Tacon et al. 2013). Indeed, there is no

transfer of carbon from C13-labelled organic matter to fruiting bodies (Le Tacon

et al. 2015). Truffle production therefore differs significantly from that of

saprotrophic fungi such as button [Agaricus bisporus (J. E. Lange) Imbach] or

oyster [Pleurotus ostreatus (Jacq.) P. Kumm.] mushrooms. Climatic changes such

as drought could affect these two bottlenecks in truffle production, and as such,

truffle growers have prioritised overcoming the negative effects of drought by

applying new management practices (see Chap. 10). Primary interventions realised

by truffle growers include soil tilling, tree pruning, irrigation and spore inoculation

(Olivier et al. 2012). As indicated in Fig. 2.2, the adaptation capacities of

T. melanosporum could also be important to overcome stresses.



Fig. 2.2 Tuber melanosporum production process. The two main bottlenecks are indicated. The

unknown and negative effects of drought on bottlenecks 1 and 2 are indicated. To counterbalance

the positive effects of cultural practices is indicated, while truffle genetic adaptation capacity is

currently unknown



24



2.2.3



J. Chen et al.



Tuber melanosporum Production in the Context

of Climate Change: The Importance of Adaptation

Capacities



Species can respond to climate change by migration and/or adaptation (Davis and

Shaw 2001). In the future, evolutionary responses will likely be critical as habitat

fragmentation impedes migration or if suitable habitats are already occupied

(Shigesada and Kawasaki 1997). Genetic variability in traits under selection,

DNA decay (accumulation of mutations in genes) and reproduction modes are

critical for adaptive evolution in response to climate change. Indeed, it seems that

genetic variance can enhance the persistence of populations in a changing environment (Etterson 2004). The effect of climate variability on mushroom productivity

has been reported for different fungal species, including truffles (Kauserud

et al. 2010; B€

untgen et al. 2011, 2012a, b). B€untgen and colleagues (2012a)

found a boost in ECM fungi harvested in their 30-year survey that may be explained

as increases in the growth of mycelium and of host plants, both of which are factors

resulting from recent climatic changes. Interestingly, this increase contradicts the

decrease of T. melanosporum fruiting bodies harvested in the twentieth century.

Indeed, it seems that drought could be responsible for the reduction of mycelial

growth, as well as the diminution of ascomata formation (B€untgen et al. 2012a, b).

B€untgen and colleagues (2012a, b) proposed that T. melanosporum would migrate

to northern ecosystems to escape the desiccating Mediterranean climate. The

authors evoked the possibility that T. melanosporum could adapt to these new

climatic conditions. Does it mean that T. melanosporum will disappear from

southern territories? The production of T. melanosporum is therefore directly linked

to the management of truffle orchards (see above) and adaptation capacities that

result from its genetic diversity and reproductive mode.

For many years, T. melanosporum was considered a selfing species with a low

level of genetic diversity (Bertault et al. 1998). The recent discovery of a heterothallic reproductive mode of T. melanosporum (Rubini et al. 2011), and small-scale

spatial genetic analyses realised with microsatellites and mating-type (MAT)

markers, suggested that T. melanosporum invests mainly in sexual reproduction

(Murat et al. 2013). Thanks to the sequencing of its genome (Martin et al. 2010), the

development of highly polymorphic microsatellite markers provides a better idea of

the real genetic diversity of this species. The genotypic diversity index, close to its

theoretical maximum of 1, suggested that almost all truffles analysed were genetically different from each other (Murat et al. 2011). A recent investigation of the

small-scale genetic structure highlighted the fact that T. melanosporum truffle

orchards are dynamic ecosystems that can contain up to 13 small genets in 30 m2

(Murat et al. 2013). Finally, the genome resequencing of six geographic accessions

from France, Italy and Spain identified a total of 442,326 single nucleotide polymorphisms (SNPs) corresponding to 3540 SNPs/Mbps, confirming that

T. melanosporum has a genetic diversity similar to those of other filamentous

fungi (Payen et al. 2015). The SNPs were more frequent in repeated sequences



2 The Black Truffles Tuber melanosporum and Tuber indicum



25



(85 %), but it was possible to identify 4501 SNPs in the coding regions of 2587

genes. This study allowed for the identification of putative genes and genomic

regions subjected to positive or negative selection and questioned the adaptation

capacities of T. melanosporum. Further research is now being conducted at INRA

Nancy to specifically address the adaptation of T. melanosporum to environmental

stresses.



2.2.4



Tuber melanosporum Biogeography



Tuber melanosporum biogeography was investigated with different molecular

markers such as randomly amplified polymorphism DNA (RAPD), microsatellites,

ITS sequencing and inter-simple sequence repeats (ISSR) (Bertault et al. 1998;

Murat et al. 2004; Riccioni et al. 2008; Garcı´a-Cunchillos et al. 2014). These

studies highlighted an important genetic structure of T. melanosporum in natural

populations mainly resulting from the effects of the last glaciation. Two putative

postglacial recolonisation routes have been hypothesised for France: one through

the west and another through the east (Murat et al. 2004). The existence of glacial

refuges was suggested in Italy and Spain (Murat et al. 2004; Riccioni et al. 2008;

Garcı´a-Cunchillos et al. 2014). Recently, Payen and colleagues (2015) estimated

that the 60,507 SNPs present in the genomic regions free of selection pressure

accumulated between 100,000 and 150,000 years ago. To be sure, this time

estimation should be considered with caution; but it still confirms that the last

glaciation, which occurred about 120,000 to 11,000 years ago (Van Andel and

Tzedakis 1996), has impacted the actual T. melanosporum genetic diversity and

genetic structure. As a result, the most recent common ancestor (MRCA) of all

geographic accession probably occurred before the last glaciation. The sampling

was not sufficient to definitively draw a conclusion on T. melanosporum history, but

it confirms previous conclusions (Murat et al. 2004). This first population genomic

analysis highlights the power of genome resequencing to investigate T.

melanosporum biogeography.



2.3



Tuber indicum: A Complex of Cryptic Species?



The Asian black truffle T. indicum was first described from a dried sample

harvested in January 1892 by Duthie near the Indian city of Mussooree (now

Mussoorie), in the northwestern Himalaya at an altitude of about 2000 m AMSL

(Cooke and Massee 1892). Much later, other similar specimens have been described

and named as different species: Tuber sinense (Tao et al. 1989), Tuber himalayense

(Zhang and Minter 1988), Tuber pseudohimalayense (Moreno et al. 1997) and

Tuber formosanum (Hu 1992). Specimens have also been described in Japan

(Kinoshita et al. 2011). The morphological variations supposed to occur between



26



J. Chen et al.



these different specimens, often observed in one or at most a few individuals, could

be considered as usual variations within a single species. This was in fact confirmed

by molecular analysis, and it is now agreed that these different taxa are synonymous

and form a single species, T. indicum, with different populations, groups, ecotypes

or cryptic species (Zhang et al. 2005; Wang et al. 2006; Chen et al. 2011; Kinoshita

et al. 2011). However, Chen et al. (2011) considered that T. pseudoexcavatum and

T. pseudohimalayense are synonymous. This discrepancy is due to taxon sampling

or attribution. Samples described as T. pseudohimalayense belonged either to T.

pseudoexcavatum or to T. indicum. Based on host plants, geographic distribution

and minor morphological differences, Chen et al. (2011) considered that

T. formosanum is a separate species from T. indicum. However, some Tuber

specimens collected from Japan displayed a close phylogenetic relationship with

Chinese T. indicum and Taiwanese T. formosanum with more than 98 % ITS

similarities with both species (Kinoshita et al. 2011), and all of them belong to

the Melanosporum group.

In China, T. indicum is found mainly in the provinces of Yunnan and Sichuan

between 25 and 30 of latitude north. Based on ITS-RFLP or ITS sequences, Roux

et al. (1999), Paolocci et al. (1997) and Zhang et al. (2005) distinguished two groups

inside the T. indicum complex. There were, however, some discrepancies among

the three studies. Wang et al. (2006) obtained results congruent with those of Zhang

et al. (2005): group I consisted of samples harvested in Huili and Huidong,

including those harvested in the South near Chuxiong and Kunming; group II

comprised all the samples harvested in Gongshan, Panzhihua, Miyi and Huize.

The genetic distances between the Chinese populations based on ITS and β-tubulin

sequences confirmed the existence of these two groups. Moreover, there was a

significant phylogeographical structure within the Chinese T. indicum complex

(Wang et al. 2006). The existence of a sinuous limit between the two groups

suggested that at least two factors could be involved in their differentiation: a

northward migration after the last glaciation and a possible recolonisation from

the bottom of the valleys. Chen et al. (2011) also showed that the Chinese

T. indicum specimens were distributed in two significant clades using the multigene

phylogenetic analysis and supposed that they should be at least two cryptic species.

Recently, the mating-type genes of T. indicum were described (Belfiori

et al. 2013). Similar to T. melanosporum, T. indicum displays only one matingtype gene per haploid genome, suggesting it is also a heterothallic species. By

analysing 115 ascomata imported to Italy from China, Belfiori et al. (2013) found

two genetic groups according to ITS sequences called A and B, with the B group

being divided into B1 and B2 as in Paolocci et al. (1997).

The sequence and organisation of the mating-type genes and idiomorphs showed

significant divergence between T. indicum truffles displaying the ITS class A and

those displaying classes B1 and B2. This result suggested the presence of at least

two cryptic species in T. indicum corresponding to T. indicum_A and T. indicum_B

(Belfiori et al. 2013). The use of mating-type genes at a large scale could therefore

enhance our understanding of the T. indicum complex.



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