1. New considerations of Tuber melanosporum life cycle
Xavier Vilanova1
, Marcos Morcillo1
, Mónica Sánchez1
, Herminia De la Varga1
1
Micologia Forestal & Aplicada. Camí d’Alfou sn, 08459 Sant Antoni de Vilamajor, Barcelona, Spain
micofora.com
corresponding author: xaviervilanova@micofora.com
Introduction
The black truffle, Tuber melanosporum Vittad., is an ascomycete fungi that produces ectomycorrhizal
associations with different trees and shrubs (Mello et al. 2006). The black Périgord truffle is known by the
organoleptic properties of their fructifications, that are naturally harvested under mediterranean regions,
but also cultivated all over the world in orchards planted with inoculated seedlings. Since 2015 it is
considered as a perennial culture for the European Union, although the production of inoculated seedlings
is done since the 70s (Murat 2015), and thus the importance of study the species. After the publication of
T. melanosporum genome (Martin et al. 2010), in the last years a huge progress has been done to unravel
truffle life cycle (Le Tacon et al. 2016, Taschen et al. 2016, De la Varga et al. 2017). Nowadays we have
more information about how the fungus live, in symbiosis with plants, and in soil. We have information
coming from scientific research, from experimental truffle sites, wild forests and established truffle
plantations.
Regarding research publications, we have an approximation to T. melanosporum life cycle. It is a
heterotallic specie, mating is controlled by two idiomorphs called MAT 1 and MAT2 (Rubini et al. 2011).
Truffles are the result of sexual reproduction, we know that a female individual (that can be either MAT 1
or MAT 2), found always as mycorrhiza, has to meet with a male one of the opposite mating type (whose
origin can be different). Most of individuals acting as fathers come from germinating ascospores, as its
principal function is the sexual reproduction (De la Varga et al. 2017, Taschen et al. 2016).
In different publications, it has been described that it exists, at root level, a kind of progressive exclusion
of the female individuals harboring the mating type opposite to the dominant one, leading to have
plantations with a patchy distribution of the mating types at root level, having each tree roots with
individuals of only one mating type (Murat et al. 2013, De la Varga et al. 2017).
One of the practices that is widespread among truffle growers, specially in Spain, is to make the called
“truffle traps”. This technique consists on doing holes or trenches around the trees and to introduce a
substrate with a mixture of spores. In those substrates, the spores come from a mixture of different
truffles, so a mixture of both mating types. In a recent publication (Murat et al. 2016) it was tested the
efficiency of this technique and showed how truffle production could be promoted and localized in a few
centimeters, opening also the possibility to better understand the role of ascospores.
Discussion
In our experiences along the years, when we observe the behavior of the spores throughout the different
steps in the process of truffle production in managed plantations (plant production, plantation
establishment and management techniques – substrate addition to increase truffle production – ) we have
seen that:

2. 1) For the production of T. melanosporum mycorrhized seedlings, the inoculation takes place in
march-april (in the northern hemisphere) observing the first mycorrhizae in October-November
(observing the roots under a microscope). Moreover, using molecular techniques, as the Real-
Time PCR (Parladé et al. 2013) it can be detected an increase of germinated mycelia, coming
from the spores, from July. (unpublished data)
2) Periodically, inoculated seedlings are analyzed to check the level of mycorrhization of the roots.
When doing those analyses in nursery plants, but also in two years old trees, that have been
planted in the field, non-germinated spores are detected. They remain in the substrate, near the
roots, and apparently, they are viable spores.
3) As it has been noticed in field experiences (data not shown) and in other publications (Murat et
al. 2016), in the places where substrate has been applied truffles fructify 2 years after the
application. The results of adding spores to the soil surrounding the trees are reflected as an
increase of the production of truffle ascocarps, that are localized in the place where the substrate
has been applied (Murat et al. 2016).
All those evidences make us hypothesize that the first germinating spores form the ectomycorrhiza, so
that they could be “predetermined” to act as mother/female individuals, or they “choose” to germinate
and act as mother/female elements and form the ectomycorrhizae. The other spores remain in the soil
waiting to germinate next year or later. This could also explains why the results of applying substrates
with spores are not seen after 2 years. Indeed, it has been reported the fructification of truffles in
plantations with inoculated trees of two years old (data not shown). In this case, it could be explained by
the germination of the spores that remained in the substrate, and that they act as male elements for the
sexual reproduction.
This simple hypothesis could explain the behavior observed in the spores at the different stages of truffle
life cycle. Currently, the mechanisms that determine that one individual acts as female or male element
are unknown (genetics, environmental, molecular, etc.), but probably this function is determined. It could
be that in the genome of each individual it was determined so that the first spores to germinate act as
female element, that could entail an energy saving for the fungus, as well as an explanation to the rates of
consanguinity described in some publications (Taschen et al. 2016).
The fact that T. melanosporum fructifies in traps with added spores after 2 years, and in young two years
old trees planted in orchards, could be explained by this hypothesis. Both kind of fructifications could be
promoted by the late germination of the spores that will act as male elements. In the example of truffle
traps these male partners will come from the spores added to the substrate; for the young seedlings, they
may come from the bank of spores remaining in the substrate of the plant. The question is that if this is
like that, we should expect to have fructification in older plants maintained in nurseries. This has never
been reported, probably the germination of the ascospores that will act as male elements is triggered by
environmental signals and that is why they remain non-germinated as a bank of spores.
Conclusion
The last years research has advanced to unravel how is the life cycle of the black truffle (Murat et al.
2013, Le Tacon et al. 2016, Taschen et al. 2016 and De la Varga et al. 2017), but there are still some
points to be clarified. More research needs to be done to explain how and why one individual act as
female or male element, especially how this is regulated. Those findings would have an impact in truffle
industry, as methodologies to promote mating will lets us to have more control on the production of
truffle ascocarps.

3. References
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genotypes in Périgord black truffle (Tuber melanosporum Vittad.) revealed contrasted reproduction
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