10.6092/issn.2531-7342/13052
Several studies focused on Tuber magnatum, the most valuable truffle species, with a limited geographical distribution. However, no comprehensive information on the thickness of its peridium (the external surface of fruiting bodies) is available. Accordingly, to fill this lack of information and in order to provide a more in-depth morphological characterisation of white truffle populations from Molise region (one of the Italian richest areas of truffles), the peridium thickness of twenty-one T. magnatum ascomata collected from two different study sites was measured by light microscope observations. A considerable variability within the analysed populations emerged, with values ranging from 271.25 µm (minimum) to 1231.25 µm (maximum), and an average peridium thickness of 622.33 µm. Interestingly, significant differences were observed between the two groups, with truffles harvested in an inner area of the region showing a peridium significantly thinner than those collected on the border with Abruzzo region. No linear correlation between peridium thickness and other morphological parameters (ascoma weight and maturity) emerged. It is likely that the differences observed between the two T. magnatum populations could be related to factors such as soil characteristics, site-specific features, genetic traits, as well as truffle collection period, which should be properly investigated.
 
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Determination of the peridium thickness of <em> Tuber magnatum </em> ascomata from Molise region.
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Zambonelli A, Rivetti C, Percudani R, Ottonello S (2000) TuberKey: a delta-based tool for the description and interactive identification of truffles. Mycotaxon 74:57-76.
Determination of the peridium thickness of <em> Tuber magnatum </em> ascomata from Molise region.
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                 IJM - Italian Journal of Mycology
                 ISSN 2531-7342 - Vol. 50 (2021): 92-98
                 Journal homepage: https://italianmycology.unibo.it/

Short note

                     Determination of the peridium thickness
                of Tuber magnatum ascomata from Molise region

         Pamela Monaco1, Gino Naclerio1, Antonio Bucci1*†, Antonietta Mello2*†

1 University of Molise, Department of Biosciences and Territory, Contrada Fonte Lappone, 86090 Pesche (IS), Italy
2 Institute for Sustainable Plant Protection (IPSP), Turin Unit, National Research Council, Viale P.A. Mattioli 25, 10125
Turin, Italy

* Corresponding author e-mail: antonio.bucci@unimol.it, antonietta.mello@ipsp.cnr.it

† These authors contributed equally as senior authors




Article info
Received 7/4/2021; accepted 7/7/2021
https://doi.org/10.6092/issn.2531-7342/13052

Abstract
Several studies focused on Tuber magnatum, the most valuable truffle species, with a limited geographical distribution.
However, no comprehensive information on the thickness of its peridium (the external surface of fruiting bodies) is available.
Accordingly, to fill this lack of information and in order to provide a more in-depth morphological characterisation of
white truffle populations from Molise region (one of the Italian richest areas of truffles), the peridium thickness of twenty-
one T. magnatum ascomata collected from two different study sites was measured by light microscope observations. A
considerable variability within the analysed populations emerged, with values ranging from 271.25 µm (minimum) to
1231.25 µm (maximum), and an average peridium thickness of 622.33 µm. Interestingly, significant differences were
observed between the two groups, with truffles harvested in an inner area of the region showing a peridium significantly
thinner than those collected on the border with Abruzzo region. No linear correlation between peridium thickness and
other morphological parameters (ascoma weight and maturity) emerged. It is likely that the differences observed between
the two T. magnatum populations could be related to factors such as soil characteristics, site-specific features, genetic
traits, as well as truffle collection period, which should be properly investigated.

Keywords
white truffle; fruiting body; external surface; morphological traits; light microscope; Central-Southern Italy


Introduction
Tuber magnatum Picco is an ectomycorrhizal ascomycete of the Pezizales order that, in symbiosis
with several trees and shrubs, produces edible hypogeous fruiting bodies, the so-called Italian white
truffles, one of the most expensive foods in the world (thousands of euros per kilogram; Laruccia et al.,
2020). In addition to the valuable culinary properties, the exorbitant prices of T. magnatum ascomata
reflect their low availability on the market, which is related to both the difficulties in cultivation and
the limited distribution range (Mello et al., 2006; Christopolous et al., 2013; Iotti et al., 2014; Riccioni
et al., 2016). Indeed, this prized truffle species, for a long time considered endemic to Italy, grows
spontaneously only in a few European countries (Rubini et al., 2005; Belfiori et al., 2020).

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and mantained by ABIS-AlmaDL. © 2021 The Authors. This work is licensed under CC BY 4.0
Monaco et al.                                                    Italian Journal of Mycology 50 (2021): 92-98

https://doi.org/10.6092/issn.2531-7342/13052



   Molise region (Central-Southern Italy) is one of the most productive area of the Italian peninsula.
Indeed, it is estimated that the total amount of T. magnatum truffles annually collected in the
Molise forests is between 30 and 70 quintals (https://www.agi.it/lifestyle/news/2021-01-11/molise-
diventa-patria-tartufo-10978317/). However, although the prized white truffle has been extensively
characterised from many points of view, Molise truffles have received very little scientific attention
(Monaco et al., 2020, 2021).
   Several studies focused on T. magnatum phylogeography and genetic characteristics (Mello et al.,
2005; Rubini et al., 2005; Belfiori et al., 2020; Monaco et al., 2021), associated microbial communities
(Barbieri et al., 2007, 2010; Amicucci et al., 2018; Monaco et al., 2021; Niimi et al., 2021), volatile
organic compounds (Vita et al., 2015; Niimi et al., 2021), molecular and biochemical traits (Vita et al.,
2020), ecological features (Marjanović et al., 2015), and cultivation techniques (Bach et al., 2021).
   Nevertheless, to date, very few data relating to the thickness of the peridium (the external surface
of truffle fruiting bodies) are available.
   The peridium takes on different features depending on Tuber species. In the black truffles Tuber
aestivum Vittad. and Tuber melanosporum Vittad., it is brown-black and characterised by the presence
of hard pyramidal warts; in Tuber borchii Vittad. it appears at first whitish, then greyish-yellow and
finally brown ochre in colour, quite pubescent when immature and, at maturity, glabrous, humid, and
smooth, often with darker or lighter reddish spots, whereas T. magnatum peridium is smooth, suede-
like, and adherent to the gleba (the inner part of the ascoma) (Hall et al., 2007; Angelini et al., 2016).
   This short note reports the preliminary results of peridium thickness measurements of two white
truffle populations (T. magnatum) harvested within natural truffle grounds in Molise region, with the
aim to 1) fill the lack of information on this parameter and 2) provide a more in-depth morphological
characterisation of the Molise white truffles.

Materials and Methods
Sampling
In this study, twenty-one ascomata belonging to two distinct T. magnatum populations were considered.
They were collected in two different areas of Molise region (Fig. 1). In particular, nine ascomata
(samples from 1 to 9, group 1) were harvested in November 2019 in the study site 1, between Carovilli
and Vastogirardi municipalities (Isernia province), an area highly suited to truffle collection (Paolanti
et al., 2014; https://www.agi.it/lifestyle/news/2021-01-11/molise-diventa-patria-tartufo-10978317/).
Other twelve samples (indicated with progressive numbers from 10 to 21; group 2) were collected in
January 2020 in the study site 2, a more extensive area on the border with Abruzzo region. Truffles
were found, with the help of trained dogs and expert truffle hunters, in natural orchards located
within mixed coppice woods, with a vegetation composed mainly of hazel trees (Corylus avellana
L.), Turkey oaks (Quercus cerris L.), beeches (Fagus sylvatica L.), and cornels (Cornus sanguinea
L.). Then, they were placed in polypropylene containers and transported to the laboratory under
refrigerated conditions (Monaco et al., 2021).
    Dried samples of each specimen were deposited in the herbarium at the University of Molise, Italy.

Determination of peridium thickness
Tuber magnatum ascomata were gently brushed and washed with distilled water to remove soil
residues. In order to determine the thickness of the peridium, fruiting bodies were divided in half and
cut lengthwise with a steel blade to obtain thin sections. For each truffle, five sections were considered
by taking the peridium in several points of the ascoma. In addition to the peridium, sections also
included a small portion of gleba to facilitate the subsequent determination of the peridium thickness
by microscopic observation (Fig. 2). The slices so obtained were placed on a microscope slide and
covered/wetted with distilled water before applying a coverslip. The thickness of the peridium was

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  Fig. 1 - Study area. The two collection sites are shown on the map: the study site 1 is located between Carovilli and
  Vastogirardi municipalities whereas the study site 2 covers a more extensive area on the border with Abruzzo region.

determined with the aid of a light microscope equipped with a ruler (Leica Microsystems, 10×
objective). For each fruiting body, it was measured in twenty-five different points (5 for each section),
positioning the microscope ruler perpendicularly to the section to be analysed (Fig. 2). The average
thickness of the peridium was then determined by calculating the arithmetic mean of the twenty-five
measured values.




                   Fig. 2 - Example of peridium thickness determination by microscope observation
                   of a thin truffle section also comprising a small portion of gleba. As shown by the
                   yellow arrows, for each section the thickness of the peridium was measured in five
                   points to obtain a total of 25 values for each ascoma.



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   In order to assess whether there were statistically significant differences in the peridium thickness
of the two investigated T. magnatum populations, Student’s t-test was performed. Moreover, an
analysis with the Pearson’s correlation coefficient was conducted in order to verify the possible linear
correlation between peridium thickness and ascoma weight and maturity [these two morphological
data are reported in Monaco et al. (2021) for the same samples here analysed].

Results and discussion
   Even if several works exist on the prized white truffle, to date, no comprehensive information on
the thickness of fruiting body peridium is available. Indeed, there is a lack of systematic studies aimed
at analysing this aspect. Some of the few data found in scientific literature have been reported by
Zambonelli et al. (2000), who examined this characteristic for 5 samples of T. magnatum from Emilia
Romagna region, and by Angelini and colleagues (2016), as part of a more extensive work mainly
focused on the isolation and identification of allelochemicals from Tuber ascomata. Consequently,
these Authors, having different goals, did not investigate the peridium thickness in relation to other
parameters (such as, for example, fruiting body size or maturity, truffle collection period, etc.).
   Accordingly, to fill this lack of information and in order to provide a more in-depth morphological
characterisation of the Molise white truffles, the peridium thickness of the twenty-one ascomata
collected from two different areas of the region, between November 2019 and January 2020, was
accurately measured by light microscope observations.
   The two examined T. magnatum populations were previously included in a wider research, in which
they were analysed from a morphological, genetic, and microbiological point of view, highlighting a
surprising heterogeneity between and within the investigated groups (Monaco et al., 2021). Although,
in agreement with prior investigations (Büntgen et al., 2017; Garcia-Barreda et al., 2021), no strong
linear correlation between fruiting body weight and maturity was found, truffles of the first group
were, overall, of greater dimensions and riper compared to those of the second group (Monaco et al.,
2021).
   Measurements of the fruiting body peridium carried out in this study further confirmed the
heterogeneity detected since, as shown in Table 1, a considerable variability within the analysed
populations emerged, with values ranging between 271.25 µm (minimum) and 1231.25 µm
(maximum).
   Overall, the peridium thickness of the examined Molise truffles was on average 622.33 µm. This
mean value differs from those recorded by Zambonelli et al. (2000), for samples collected in Northern
Italy, and by Angelini et al. (2016).
   Interestingly, significant differences were observed between the two investigated populations
(Student’s t-test, p < 0.01): indeed, truffles harvested between Carovilli and Vastogirardi municipalities
(group 1) showed a peridium significantly thinner than those of the group 2, with average values of
452.42 and 749.76 µm, respectively.




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                   Table 1 - Average peridium thickness of the twenty-one white truffles (T.
                   magnatum) from Molise region.

                       SAMPLE
                                    GROUP          AVERAGE PERIDIUM THICKNESS* (µm)
                        CODE
                          1             1                             298.75
                          2             1                             271.25
                          3             1                             287.68
                          4             1                             521.36
                          5             1                             487.00
                          6             1                             667.50
                          7             1                             482.22
                          8             1                             533.50
                          9             1                             522.50
                         10             2                            1231.25
                         11             2                             813.75
                         12             2                             985.16
                         13             2                             444.29
                         14             2                             798.75
                         15             2                             653.13
                         16             2                             757.50
                         17             2                             394.17
                         18             2                             637.67
                         19             2                             854.46
                         20             2                             615.91
                         21             2                             811.03

                   The average peridium thickness of each fruiting body was given by the arithmetic
                   *

                   mean of the twenty-five measured values

   The analysis with Pearson’s correlation coefficient revealed that there is no linear correlation
between truffle weight and peridium thickness (r = - 0.21) and only a slight/moderate negative
relationship between fruiting body maturity and the thickness of the peridium (r = - 0.50).
   In our opinion, it is likely that the differences observed in the peridium thickness between the two
analysed T. magnatum populations, similarly to the differences previously reported for truffle weight
and maturity (Monaco et al., 2021), could be related to the diverse collection period of the ascomata
(fruiting bodies of the first group were collected in November whereas those of the second group
in January). Indeed, it is known that the white truffles of January can be morphologically different
from T. magnatum collected between October and December, because of the ripening in winter
period, under frost and ice (http://www.artopoltrepo.it/Documenti/manualetartufi.pdf). Therefore,
we might assume that the thicker peridium of the fruiting bodies harvested in January 2020 (group
2) could represent a kind of adaptation to unfavourable environmental conditions. However, since
no comparison data are available, further analyses will be required to shed light on an effective
correlation between peridium thickness and environmental/climatic conditions. On the other hand,
the remarkable variability detected within the examined T. magnatum populations could be explained
taking into account other factors that should be properly investigated, such as genetic traits, site-
specific features, soil characteristics, and microclimatic conditions.

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Conclusion
In the future, it would be interesting to carry out further analyses including a larger number of ascomata
collected in different areas of the region and in other Italian regions, also to verify the existence of
a possible link between the peridium thickness and T. magnatum provenance. Indeed, the potential
use of the peridium thickness as an effective parameter to track the geographical origin of truffles
could have a significant impact on the economy related to this product (mainly at regional scale) from
different perspectives (truffle traceability, promotion and conservation), and represents a relevant
aspect to be properly investigated in future researches.

Acknowledgements
We are grateful to Fabio D’Amico (“San Michele” agricultural cooperative) and Giuseppe Beniamino
(“Tartufi-Le Ife” company) for their valuable contribution during truffle searching and collection.
We also thank Professor Alfredo Vizzini (University of Turin, Italy) for his helpful advice during
morphological investigations and Antonella Faccio (Institute for Sustainable Plant Protection, Turin,
Italy) for technical suggestions.

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