10.6092/issn.2531-7342/12822
Inside commercial batches of T. aestivum imported from Iran to Italy the ascomata of six Tuber spp. (Tuber borchii, Tuber brumale, Tuber macrosporum, Tuber rufum f. lucidum, Tuber excavatum and Tuber fulgens) were found and described. Sixteen specimens were analyzed; most of them were completely immature probably because truffles in Iran are harvested by digging all the soil forest without using trained dogs. The morphology of the Iranian ascomata and their spore dimensions are similar to those of the European ascomata and phylogenetic analyses based on their ITS sequences placed them inside the European clades of Tuber. In Iran, truffles are found in the northern part of the country with the highest production in the Hyrcanian region (between Guilan and Golestan provinces). To date, Iran seems to be the most Eastern location where European species of Tuber are found.
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True truffle diversity in Iran
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True truffle diversity in Iran
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 50 (2021): 52-62
                Journal homepage: https://italianmycology.unibo.it/

Original paper

                                   True truffle diversity in Iran

               Federico Puliga1, Mirko Illice2, Mirco Iotti3*, Pamela Leonardi1,
                Ali Baghdadi1, Ali Akbar Mozafari4, Alessandra Zambonelli1

1 Department of Agricultural and Food Sciences, University of Bologna, Viale G. Fanin 44, 40127, Bologna, Italy
2 AUSL of Bologna, Via Castiglione, 29 - 40124 Bologna, Italy
3 Department of Life, Health and Environmental Science, University of L’Aquila, via Vetoio, 67100, Coppito, L’Aquila,
Italy
4 Department of Horticultural Sciences, Faculty of Agriculture, University of Kurdistan, Sanandaj, Iran

* Corresponding author e-mail: mirco.iotti@univaq.it


Article info
Received 19/4/2021; accepted 2/6/2021
https://doi.org/10.6092/issn.2531-7342/12822

Abstract
Inside commercial batches of T. aestivum imported from Iran to Italy the ascomata of six Tuber spp. (Tuber borchii,
Tuber brumale, Tuber macrosporum, Tuber rufum f. lucidum, Tuber excavatum and Tuber fulgens) were found and
described. Sixteen specimens were analyzed; most of them were completely immature probably because truffles in Iran
are harvested by digging all the soil forest without using trained dogs. The morphology of the Iranian ascomata and their
spore dimensions are similar to those of the European ascomata and phylogenetic analyses based on their ITS sequences
placed them inside the European clades of Tuber. In Iran, truffles are found in the northern part of the country with the
highest production in the Hyrcanian region (between Guilan and Golestan provinces). To date, Iran seems to be the most
Eastern location where European species of Tuber are found.

Keywords
Iran, true truffles, Tuber borchii, Tuber brumale, Tuber macrosporum, Tuber rufum, Tuber excavatum, Tuber fulgens


Introduction
Truffles are generally considered to be all those fungi that form subterranean fruiting bodies and live
in a symbiotic relationship with the roots of one or more host plants. They are a large polyphyletic
group with representatives in the Glomeromycota, Ascomycota, and Basidiomycota (Læssøe et al.,
2007). However, strictly speaking “true truffles” are limited to those ascomycetes belonging to the
pan-global genus Tuber (Zambonelli et al., 2016) which comprises around 200 species worldwide
distributed (Bonito et al., 2010). There are 35 species of Tuber in Europe (Leonardi et al., 2021) but
only a few of them are highly appreciated because of their special taste and smell. In Italy, for example,
only seven species of Tuber are harvested and commercialized after the national law L.N. 752/1985:
Tuber magnatum Picco (the Italian white truffle), Tuber melanosporum Vittad. (the Perigord black
truffle), Tuber aestivum Vittad. (the summer truffle), Tuber borchii Vittad. (the bianchetto truffle),
Tuber brumale Vittad. (the winter truffle), Tuber macrosporum Vittad. (the smooth black truffle) and
Tuber mesentericum Vittad. (the ordinary truffle). Although no species in the genus are considered
poisonous some are considered inedible either because they have an unpleasant aromas, like Tuber
rufum Vittad. and Tuber maculatum Vittad. or have a hard flesh, like Tuber excavatum Vittad.

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
Puliga et al.                                                       Italian Journal of Mycology 50 (2021): 52-62

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   Overexploitation of truffle resources, climate warming, a decrease of land surface devoted to
truffles and natural habitat deterioration due to the abandoned management of the old truffle orchards
have been considered the major causes of natural truffle production decline in Europe (Hall et al.,
2007; Büntgen et al., 2012; Le Tacon et al., 2014).
   The cultivation of truffle species (T. melanosporum, T. aestivum and T. borchii) has been successfully
achieved in Europe and other continents, like Australia, New Zealand, the USA, Chile and South Africa.
Nevertheless, the availability of these truffles is still insufficient to satisfy the increasing market demand.
For this reason, truffle companies are constantly searching for new supplies. Starting around 1994 more
than 20 tons of Chinese black truffles (especially Tuber indicum Cooke & Massee) have been imported
into Europe annually (Riousset et al., 2001). In the same years, European truffles were re-discovered in
the Balkan Peninsula, especially in Hungary, after the harvesting restrictions of the communist era were
relaxed (Gógán Csorbai et al., 2007). Consequently, T. magnatum, T. aestivum and T. brumale began to
be intensively harvested and exported to other European countries, especially Italy.
   More recently, T. aestivum, as well as other Tuber species, were also found in the Caucasus and
surrounding countries such as Iran (Badalyan et al., 2005; Bagi and Fekete, 2010; Jamaly, 2017;
Ammarellou and Alvarado, 2018; http://www.mnp.am/red_book_fauna/eng/p520.html) but the
knowledge on the diversity of truffles from this area is limited particularly in Iran truffles. In recent
years Puliga and colleagues (2020) have reported that tons of T. aestivum ascomata have been imported
into Italy from Iran and while inspecting some commercial batches they found and described a new
species Tuber iranicum. Regarding the presence of T. brumale in Iran it has been reported by the
sequences of its mycorrhizas (Bahram et al., 2012), but the ascomata were not described. To the best
of our knowledge, no other species of Tuber have been reported from Iran.
   In the work, 16 ascomata from Iran morphologically similar to European Tuber species were
DNA barcoded by sequencing their ITS rDNA regions and phylogenetically compare them with
morphologically similar European species belonging to the same Tuber clade (Bonito et al., 2010;
2013). We also make morphological descriptions of their spores and report the distribution and
ecological features of the truffle areas of Iran where they are collected.

Materials and methods
The Tuber spp. ascomata analyzed in this study were found in commercial batches of T. aestivum
that had been imported from Iran to Italy in 2018 (29th August, 15th November, 7th December, 19th
December) and 2019 (16th March). A part of each ascoma was either dried and preserved in the CMI-
Unibo Herbarium of Bologna University, another portion was fixed in FAA (90% ethanol 70%, 5%
acetic acid and 5% formaldehyde) for later micro-morphological analyses and the remaining portion
was freeze-dried for 48 h in a Virtis Benchtop 2 K lyophilizer (SP Industries) and then preserved at
−20 °C pending molecular analysis.
   Each ascoma was tentatively assigned to a Tuber species on the basis of their macro-microscopic
characters (Pegler et al., 1993; Montecchi and Sarasini, 2000; Zambonelli et al., 2000; Ceruti et al.,
2003). Microscopic observations were made on gleba fragments mounted in Hoyer’s medium (Glime
and Wagner 2017; Ita Paz personal communication) using an Eclipse TE 2000-E microscope (Nikon).
Spore measurements were made by Nis-Elements AR (v 3.10) software (Zeiss) from images captured
with a DXM1200F digital camera (Nikon).
   At least 30 measurements for 3-4 spore asci, depending on the specimens, were taken (Gross,
1987; Zambonelli et al., 2000) and reported as (minimum), mean ± standard deviation (maximum).
The average spore length/width ratio (Qm) was also calculated. The degree of maturity was calculated


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on the basis of the percentage of asci having formed spores: immature 0%, partially immature 1-25%,
slightly immature 25-50%, mature 50-100%.
   For molecular analyses, DNA was extracted from 20 mg of two lyophilized ascomata using
NucleoSpin® Plant II Kit (Macherey-Nagel, Düren, Germany) following the manufacturer’s instructions.
ITS region of the rDNA was amplified using primer pairs ITS1F/ITS4 (White et al., 1990; Gardes and
Bruns, 1993). PCRs were carried out in 50-μl reaction volumes using 2x BiomixTM (Bioline) and
amplifications were run in a SimpliAmp thermal cycler (ThermoFisher) as follows: 95 °C for 6 min,
followed by 30 cycles of 94 °C for 30 s, 56 °C for 30 s. PCR products were purified using NucleoSpin®
Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany) and sequencing was performed with
both ITS forward and reverse Sequences were aligned and edited manually with BioEdit 7.0.5.3 (Hall,
1999) and compared to those deposited in GenBank (www.ncbi.nlm.nih.gov).
   Phylogenies of Iranian truffles were constructed using Maximum Likelihood (ML) and Bayesian
Inference (BI) in raxmlGUI 1.5b2 (Silvestro and Michalak 2012) and MrBayes 3.2.2 (Ronquist
et al., 2012), respectively. Sequences generated in this study were analyzed separately for each
Tuber clade (Bonito et al., 2010) using GenBank ITS sequences obtained from ascomata collected
in Europe. Tuber brumale accession MT495426 was used as an outgroup for all phylogenetic
reconstructions except for the Melanosporum clade where it was substituted by T. melanosporum
sequences (KM659870, KF591751, KM659874). Multiple sequence alignments were performed
with CLUSTALW (Thompson et al., 1994). ML analyses were performed with 1,000 throughout
bootstrap replicates (100 runs) and applying the GTRGAMMA model of nucleotide substitution for
phylogenetic reconstructions in Puberulum, Melanosporum, Excavatum and Macrosporum clades
and GTRCATI for Rufum clade. BI analyses were conducted using the best fit-models of nucleotide
substitution selected by JModelTest v2.1.4 (Darriba et al., 2012) under the Akaike information
Criterion (Puberulum and Melanosporum: HKY+G; Excavatum: TrNef+G; Macrosporum: TIM2+G;
Rufum: TIM2+I). To ensure convergence, two independent MCMC runs with six chains each were
performed in parallel for 5,000,000 generations, sampling every 100 generations. A majority-rule
consensus tree was generated after the exclusion of the first 250 trees and the posterior probabilities
of each node were computed. ML and BI trees were edited using FigTree v1.4.3 (http://tree.bio.ed.ac.
uk/software/figtree/).
   The distribution of truffles in Iran was sourced from Ali Baghdadi and Ali akbar Mozafari by
interviewing local harvesters and traders. They also provided the climate characteristics of the areas.

Results and discussion
Morphological analyses
On the basis of the descriptions available in the literature (Pegler et al., 1993; Montecchi and Sarasini,
2000; Zambonelli et al., 2000; Ceruti et al., 2003) the 16 representative ascomata of truffles purporting
to be Tuber aestivum were found to be T. borchii, T. brumale, T. rufum f. lucidum, T. macrosporum,
T. excavatum and Tuber fulgens Quél. (Figs 1 and 2). The main difference between the imported T.
brumale and Italian specimens was that two out of four ascomata had a basal cavity which is rarely
found in Italian specimens (Montecchi and Sarasini, 2000; Zambonelli et al., 2000; Ceruti et al.,
2001) and more akin to French specimens (Riousset et al., 2001).
   The micro-morphological characteristics of the analyzed truffles are reported in table 1 and
Figures 1 and 2. Seven ascomata were immature and the asci empty of spores probably because
truffle harvesting in Iran is carried out by excavating the ground as if digging for potatoes without
using trained dogs (Puliga et al., 2020). As a consequence, to confirm the macro morphological
identification, we had to resort to DNA barcoding (Supplementary Figs 1-5).

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            Fig. 1 – Ascomata (a) and spores of immature (b) and partially mature (c and d) T. borchii n. 5005,
            5006, 5007 and 5008. Ascomata (e) and spores (f) of T. brumale n. 5000 and 5001. Ascomata (g)
            and spores (h) of T. macrosporum n. 4937 and 4038. Bars = 20 µm.


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            Fig. 2 – Ascoma (a) and spores (b) of Tuber rufum f. lucidum n. 4944. Immature ascomata of
            Tuber excavatum (c) n. 5036 and Tuber fulgens (d) n. 5034. Bar = 20 µm.



    It was not always possible to make spore measurements in 4-spored asci in the mature specimens
because T. borchii 5007 and the two T. macrospoum ascomata had only a maximum of 3-spored asci.
The spore dimensions of all Tuber species were similar to those previously reported by Zambonelli et
al. (2000). Only T. macrosporum spores appeared slightly smaller than those measured by Zambonelli
et al. (2000) and Ceruti et al. (2003) but similar to those of Montecchi and Sarasini (2000). Although
the spores of T. brumale 4967 in 4-spored asci were similar or slightly smaller than those of the
other Iranian T. brumale specimens and congruent with the measurements reported in the literature
(Montecchi and Sarasini, 2000; Zambonelli et al., 2000), this ascoma had spores up to 55 µm long in
1-spored asci (data not shown). It is known that spore dimensions are inversely proportional to the
number of spores per ascus (Gross, 1987) and the anomalous abundance of one-spored asci may have
favored the formation of exceptionally large spores. The unusual number of spores per ascus is not
surprising due to its high variability even in the ascomata of a single species (Weden et al., 2005).




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Table 1 – Morphological characteristics and GenBank accession numbers of the Tuber ascomata found inside commercial
batches of T. aestivum from Iran.

     CMI-                                                                                    spore measures                                       Orn..   Accession
     Unibo            species          Ascoma characteristics      arrival date                                             spore/ascus     QM
                                                                                                  µm1                                             µm       number
      n.

    4980     Tuber borchii             immature                   15/11/2018      n.a.                                     n.a.           n.a.   n.a.     MW829424

    5005     Tuber borchii             immature                   07/12/2018      n.a.                                     n.a.           n.a.   n.a.     MW829427

    5006     Tuber borchii             immature                   07/12/2018      n.a.                                     n.a.           n.a.   n.a.     MW829425

    5007     Tuber borchii             partially immature         19/12/2018      (24) 28- 39 (45) x (18) 22-28 (34)2 1-3                 1.3    2-5      MW829423

    5008     Tuber borchii             slightly immature          19/12/2018      (24) 27- 33 (36) x (20) 21-25 (27)       1-4 (5)        1.3    2-4      MW829426

    5047     Tuber borchii             mature                     16/03/2019      (22) 27- 34 (39) x (21) 24-28 (30)       1-3 (4)        1.2    3-6      MW829422

    4946     Tuber brumale             immature                   28/08/2018      n.a.                                     n.a.           n.a.   n.a.     MW829418

    4967     Tuber brumale             mature                     15/11/2018      (20) 24- 30 (33) x (13) 15-18 (19)       1-4 (5)        1.6    3-6      MW829421

    5000     Tuber brumale             Mature, with a basal cavity 07/12/2018     (22) 27- 32 (34) x (14) 16-18 (20)       (2-3) 4-6      1.7    3-5      MW829420

    5001     Tuber brumale             Mature, with a basal cavity 07/12/2018     (22) 26- 34 (36) x (13) 16-18 (20)       (2-3) 4-6      1.8    3-10     MW829419

    4937     Tuber macrosporum         mature                     29/08/2018      (32) 41- 51 (55) x (21) 24-29 (32) 2 1-3                1.7    2-6      MW884553

    4938     Tuber macrosporum         mature                     29/08/2018      (39) 42- 49 (54) x (22) 24-28 (29)   2
                                                                                                                           1-3            1.7    3-7      MW884554

    4944     Tuber rufum f. lucidum    slightly immature          29/08/2018             (19) 21- 26 (28) x 16-19 (22) 1-4                1.3    2-5      MW829417

    5036     Tuber excavatum           immature                   16/03/2019      n.a.                                     n.a.           n.a.   n.a.     MW884550

    5034     Tuber fulgens             immature                   16/03/2019      n.a.                                     n.a.           n.a.   n.a.     MW884551

    5035     Tuber fulgens             immature                   16/03/2019      n.a.                                     n.a.           n.a.   n.a.     MW884552



1
    The spore measures were taken in 4-spore-asci with the exception of the ascomata n. 5007, 4937 and 4938. n.a. = not applicable
2
    Spore measures taken in 3-spore-asci




Molecular analyses
Molecular analysis confirmed morphological identifications of the examined specimens. Phylogenetic
analyses clustered all Tuber specimens from Iran in clades formed by European species (Supplementary
Figs 1‒5).
   The Iranian sequences of the ascomata 4980, 5005, 5006, 5007, 5008 and 5047 belong to T. borchii
and all clustered with those of the haplotype I identified by Bonuso et al. (2010) (Supplementary Fig.
1). The specimen 4944 classified as T. rufum f. lucidum for the dark color of the peridium (Montecchi
and Sarasini, 2000) falls within the “Tuber lucidum complex” clade defined by Healy et al. (2016)
(Supplementary Fig. 2).
   Although morphological differences were found between the four Iranian T. brumale ascomata
examined, genetic analyses showed the absence of genetic variability in their ITS regions
(Supplementary Fig. 3). All the specimens (4946, 4967, 5000, 5001) morphologically classified as
T. brumale clustered together with the sequences of Haplogroup II of T. brumale after Merényi et al.
(2014; 2016). This haplogroup comprises ascomata from Eastern Europe as well as other 2 accessions
from Iran (FR852065 and FR852067).
   No nucleotide polymorphisms were found in ITS sequences of the specimens 4937 and 4938,
which did not show evident morphological differences with respect to European T. macrosporum.
They seem to belong to the clade I in T. macrosporum identified by Benucci et al. (2016) althought
their sequences form a separated subclade poorly supported by both bootstrap value and posterior
probability. Deeper molecular investigations will be needed to define the phylogenetic relationships
between European and Iranian T. macrosporum (Supplementary Fig. 4).
   Molecular analysis confirmed also the morphological identifications of ascomata attributed to the

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Excavatum clade. The specimen 5036 belongs to the cryptic species of T. excavatum identified as
clade III after Puliga et al. (2020) while the ascomata 5034 and 5035 fall among T. fulgens specimens.
It is surprising the diversity of Iranian Tuber species inside the Excavatum clade. Until now, three
European species have been already described (T. fulgens, T. excavatum and T. iranicum) and other
cryptic species could be found with a more in-depth study of Iranian truffles (Supplementary Fig. 5).

Distribution and ecology
Iran seems to be the most Eastern location of European native Tuber species. Although it was not
possible to know the exact collection localities of the Iranian truffles analysed in this work, they
should have be harvested in the same localities where T. aestivum is found. Tuber aestivum were first
found in the eastern part of Iran in the Tangrah region but the highest truffle production is concentrated
in the Hyrcanian region (from Guilan to Golestan provinces) (Hamza, 1994) (Fig. 3). In these areas,
T. aestivum is mostly found in sandy high pH soils with a high CaCO3 content, and containing a low
amount of organic matter (Jamali, 2017).




  Fig. 3. Distribution and productivity of truffles in the Northern parts of Iran (a). Typical truffle producing forests (b
  and c).

   It grows especially in broad leaved forests populated by Quercus infectoria Oliv. and also other
oak species (Quercus brantii Lindl., Quercus castaneifolia C. A. Mey, Quercus macranthera Fisch.
& C.A. Mey) Persian ironwood (Parrotia persica (DC.) C.A. Mey), medlar (Mespilus germanica L.),
whitethorn (Crataegus oxycantha L.), hawthorn (Crataegus pontica K. Koch), and hazelnut (Corylus
avellana L.).
   The highest truffle presence (Fig. 3) is concentrated in the central areas of Golestan province
at 37°N and 55°E, at 150-500 m above sea level. This region consists of the mountain and plain
area. Regarding data from meteorological stations of the region over the period 2010–2020 (IRIMO,
2021), the climate is humid temperate, the highest, lowest and average annual temperatures are +40.4
°C, -9.2 °C and +17.58 °C, respectively. Furthermore, the average annual rainfall is about 676 mm.
The maximum relative humidity is 85.8%, minimum 23.8% and the annual average is 68.2%. The

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maximum daily rainfall is 90 mm and the number of rainy days is 59 days/y. The average of number
of frost days is 24 days/y and the average of daily sunny hours is 5.49 h (Supplementary Fig. 6a).
    Good truffle productions have been also reported in southern and western regions of Golestan
province at 36°N and 54°E, at 140 m above sea level. The climate in these areas is humid temperate
and the maximum daily rainfall is 65.2 mm and also the average annual rainfall is 350 mm. The
highest, lowest and average annual temperatures are +44 °C, -12 °C and +17.98 °C, respectively.
In addition, the number of frost days is 12.7 days/y and the average of daily sunny hours is 7.38 h.
The maximum relative humidity is 99.6%, the minimum 11.6% and the annual average is 59.26%
(Supplementary Fig. 6b).
    In the central and eastern part of Mazandaran province at 36°N and 53°E, at 50-120 m above sea
level, truffle production is low; the maximum relative humidity is 98.6%, minimum 41.6% and the
annual average is 74.56%. The average annual rainfall is up 423 mm and the maximum daily rainfall
is 73 mm. The highest, lowest and average annual temperatures are +39.6 °C, -8.4 °C and +17.67 °C,
respectively. The average number of frost days is 16.6 days/y and the average of daily sunny hours is
4.14 h (Supplementary Fig. 6c).

Conclusions
This work provides new insights into the truffle diversity of Iran near the previously reported T.
aestivum site (Jamaly, 2017; Ammarellou and Alvarado, 2018), and T. iranicum (Puliga et al., 2020),
another six European Tuber species from Iran are described. Three of them, T. borchii, T. macrosporum
and T. brumale are economically important for their gastronomic qualities; that opens the possibility
of developing the commercial harvesting and trade of also these truffle species in Iran.
   However, in order to preserve this important resource, it is important that proper truffle harvesting
rules will be introduced in order to avoid overexploitation that is a common problem in the countries
where truffle resources have only were recently been discovered (Chen et al., 2018). The introduction
of trained dogs is mandatory because they detect only mature truffles with the typical aroma. The use
of spades, hoes or other inappropriate tools to collect truffles represents a waste of valuable resources
as well as a serious threat to conservation of natural habitats. It should be avoided the same was done
in China in the 1980s for the matsutake areas where inexperienced Chinese collectors used rakes or
small adzes to remove the litter layer, dig through the topsoil and find the priced grade 1 matsutake
[Tricholoma matsutake (S. Ito et Imai) Sing]. This process caused irreparable damage not only to the
shiros but also to the understory vegetation and soil structure (Ian R. Hall, personal communication).
The sustainable utilization of natural resources, such as the edible ectomycorrhizal mushrooms,
should be imperative in the modern society to protect our forests and environment.

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