10.6092/issn.2531-7342/15348
Tuber borchii is a precious truffle, which forms ectomycorrhizal associations with several coniferous and broadleaf trees, arbutoid mycorrhizas with the strawberry tree and recently it was found also to form orchid mycorrhizas with Epipactis atrorubens. In this work, we investigated the formation of T. borchii mycorrhizas in other orchid species (Anacamptis morio, Cephalanthera longifolia, Limodorum abortivum, Ophrys sphegodes, Orchis purpurea). To this aim the orchids growing in three T. borchii production areas located in Emilia Romagna region (Italy) were collected and the presence of T. borchii mycorrhizas was investigated by morphological and molecular approaches. For T. borchii molecular identification, specific primers were used by a nested PCR approach. Tuber borchii was identified in 2 out of 6 samples of A. morio, 3 out of 4 samples of C. longifolia, and 2 out 6 samples of O. sphegodes. The ecological and biological roles of the orchid association in the truffle life cycle are discussed.
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Zambonelli A, Iotti M, Boutahir S, Lancellotti E, Perini C, Pacioni G (2012) Ectomycorrhizal fungal communities of edible ectomycorrhizal mushrooms. In: Edible Ectomycorrhizal Mushrooms: Current Knowledge and Future Prospects (Zambonelli A., Bonito GM, eds), Soil Biology, vol 34. Springer-Verlag, Berlin, pp. 105–124. https://doi.org/10.1007/978-3-642-33823-6_7
Symbiotic interactions between orchids and <em>Tuber borchii</em>
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                IJM - Italian Journal of Mycology
                ISSN 2531-7342 - Vol. 51 (2022): 58-65
                Journal homepage: https://italianmycology.unibo.it/

Short note

       Symbiotic interactions between orchids and Tuber borchii

                  Simone Graziosi, Pamela Leonardi, Alessandra Zambonelli
Department of Agricultural and Food Sciences, University of Bologna, Viale Fanin 44, 40127 Bologna, Italy

Corresponding author e-mail: simone.graziosi5@unibo.it


Article info
Received 11/08/2022; accepted 6/10/2022
https://doi.org/10.6092/issn.2531-7342/15348

Abstract
Tuber borchii is a precious truffle, which forms ectomycorrhizal associations with several coniferous and
broadleaf trees, arbutoid mycorrhizas with the strawberry tree and recently it was found also to form orchid
mycorrhizas with Epipactis atrorubens. In this work, we investigated the formation of T. borchii mycorrhizas
in other orchid species (Anacamptis morio, Cephalanthera longifolia, Limodorum abortivum, Ophrys
sphegodes, Orchis purpurea). To this aim the orchids growing in three T. borchii production areas located in
Emilia Romagna region (Italy) were collected and the presence of T. borchii mycorrhizas was investigated
by morphological and molecular approaches. For T. borchii molecular identification, specific primers were
used by a nested PCR approach. Tuber borchii was identified in 2 out of 6 samples of A. morio, 3 out of 4
samples of C. longifolia, and 2 out 6 samples of O. sphegodes. The ecological and biological roles of the orchid
association in the truffle life cycle are discussed.

Keywords
Bianchetto truffle, Anacamptis morio, Cephalanthera longifolia, Limodorum abortivum, Ophrys sphegodes,
Orchis purpurea, mycorrhiza



Introduction

True truffles are all species of hypogeous fungi into the genus Tuber of the family Pezizaceae, phylum
Ascomycota. They include around 200 species, but only a few of them form ascomata of considerable
gastronomic and economic value. The European species Tuber magnatum Picco, Tuber melanosporum
Vittad., Tuber aestivum Vittad. and Tuber borchii Vittad. are the most sought-after and command
prices ranging from 30 €/Kg to 6000 €/kg depending on species, ascoma size, season and availability.
Tuber borchii, commonly called “bianchetto”, is a truffle with a broad ecological adaptability and it
naturally grows in a wide geographic area from Finland to Sicily and from Portugal to Iran (Hall et
al., 2007; Bajaj and Shamekh, 2021; Puliga et al., 2021). Although it prefers sandy, calcareous soils, it
can be found also in clay and sub-acidic soils (Zambonelli et al., 2002; Gardin 2005; Hall et al., 2007;
Lancellotti et al., 2016). As all the other Tuber spp., it forms ectomycorrhizas (ECM) with a broader
range of host plants, including several shrubs and both coniferous and broadleaf trees. Moreover, it
was recently found to form arbutoid mycorrhizas with strawberry tree (Arbutus unedo L.; Lancellotti
et al., 2014) and to be associated with the mixotrophic orchid Epipactis atrorubens (Hoffm.) Besser
(Tešitelová et al., 2012).

Department of Agricultural and Food Science in partnership with the Italian Mycological Society (UMI).
The journal is hosted and maintained by ABIS-AlmaDL. © 2022 The Authors. This work is licensed under CC BY 4.0
Graziosi et al.                                                   Italian Journal of Mycology 51 (2022): 58-65

https://doi.org/10.6092/issn.2531-7342/15348


   Other Tuber species, such as T. aestivum and Tuber maculatum Vittad. as well as other typical ECM
fungal species such as Cortinarius spp., Inocybe spp., Russula spp. and Hymenogaster spp., were
found to form mycorrhizas with several orchid species belonging to the genera Epipactis, Limodorum,
Cephalantera, Vanilla, Cypripedium (Selosse et al., 2004; Julou et al., 2005; Ogura-Tsujita and
Yukawa, 2008; Ouanphanivanh, 2008; Illyés et al., 2010; Těšitelová et al., 2012; Jacquemyn et al.,
2017; Schiebold et al., 2017; Johnson et al., 2021). The connections between orchids of the genera
Cephalanthera and Epipactis and T. aestivum are so strict that Ouanphanivanh (2008) gave a first
evidence that they could be an indicator of truffle habitat fifteen years ago.
   More recently, a virtual method to detect the T. aestivum production areas in Poland and applicable
to other European countries was perfected. It considers the presence of orchids of the genera
Cephalanthera, Cypripedium or Epipactis, in addition to several other ecological parameters (such
as soil physical and chemical characteristics, host plants, etc.), as key indicators of truffle presence
(Rosa-Gruszecka et al., 2021).
   The aim of this study is to increase the knowledge about the range of orchid species forming
symbiosis with the “bianchetto” truffle in its natural habitat in Italy. To this aim all the orchids growing
in T. borchii production areas were collected and the presence of T. borchii mycorrhizas was detected
using morphological and molecular approaches.

Materials and methods

Site of study and sampling
Three T. borchii production areas were selected as study sites (Table 1). One site is located in the
littoral area of Ravenna Province into the Parco Regionale del Delta del Po (Nature 2000 protected
area IT4070005) (Corticelli et al., 2004), which is one of the most productive areas of T. borchii in
Italy (Zambonelli et al., 2002). In these sites, the T. borchii host plants are 20-50 years old Pinus
pinaster Aiton and Pinus pinea L. trees. The other two areas are located in the Apennines-Mountains
in Bologna province. In one of this area, situated in Guzzano (Pianoro, Bologna), the putative host
plant is Pinus nigra J.F. Arnold and in the other area, located in Calderino (Bologna), the host plants
are Quercus pubescens Willd. and Corylus avellana L. The detailed characteristics of these areas are
reported in Table 1 and in Supplementary Fig. S1.
    During February and March 2021 or 2022, the study sites were visited with trained dogs in order
to detect the T. borchii production patches (Supplementary Fig. S2). The GPS coordinates of each
ascoma collection point were recorded but not given here at the request of the truffle hunters. All the
collected fruiting bodies were morphologically (Zambonelli et al., 2000) and molecularly identified
using T. borchii and Tuber dryophilum Tul. & C. Tul. specific primers (TboI-TboII and TdryI-TdryII,
Amicucci et al., 1998) by direct PCR (Iotti and Zambonelli, 2006). Tuber dryophilum was included in
the analysis because its ascomata are morphologically similar to those of T. borchii and they are often
found in the same T. borchii production areas (Zambonelli et al., 2012). The collected ascomata were
then dried and deposited in the herbarium of the “Centro di Micologia” of Bologna (CMI-UNIBO).
During the orchid flowering time (April–May) of the same year of ascoma collection all the orchids
in an area of 10 m2 around each T. borchii ascoma collection point were harvested. The roots of
each plant were washed in running water to remove soil particles and preserved at – 80 °C pending
molecular and morphological analyses.




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Table 1 - Description of sampling areas and sampling dates.

       Sampling area          Area size (m2)          Soil classification1          Putative host plants Sampling dates


    Calderino, Monte San                        Oxyaquic Haplustepts fine silty,    Quercus pubescens      29/02/20222
                                  19,900
        Pietro (BO)                               mixed, superactive, mesic          Corylus avellana      27/04/20223
                                                                                                           23/02/20212
                                                Typic Ustorthents loamy, mixed,
Guzzano, Pianoro (BO)             41,300                                                Pinus nigra        05/03/20212
                                               active, calcareous, mesic, shallow
                                                                                                           09/05/20213

                                                 Aquic Ustipsamments, mixed,          Pinus pinaster       16/03/20222
      Porto Corsini (RA)          48,000
                                                            mesic                      Pinus pinea         14/04/20223
1
    USDA (2010), from https://agri.regione.emilia-romagna.it/Suoli/; 2sampling of ascomata; 3sampling of orchids

Molecular and morphological analyses
Healthy roots of around 1 cm in length were selected from the root system of each plant under
a dissecting microscope. For molecular analyses three of these root pieces were surface sterilized
for 5 min in ethanol 70% and 15 s in sodium hypochlorite 0.9%, and rinsed three times in sterile
water following the protocol of Cao et al. (2004). This protocol was previously applied to detect the
endophyte roots colonization of T. aestivum and T. melanosporum in non-ectomycorrhizal plants
(Schneider-Maunoury et al., 2020). To facilitate cell lysis, the roots pieces were firstly added to 300
mL of PL1 buffer of the NucleoSpin® Plant II kit (Macherey-Nagel, Germany), crushed using a
micropestle in sterile sand and then disrupted with a Tissue Lyser (Qiagen, USA) for 10 min at 30
Hz. Then the DNA was extracted with the NucleoSpin® Plant II kit following the manufacturer’s
instructions.
    The presence of T. borchii and T. dryophilum inside the orchid roots was assessed through a nested
PCR approach using PCR conditions after Leonardi et al. (2021). Tuber dryophilum was included
in the analysis when its ascomata were found in the same T. borchii production patches (see results
section). The fungal DNA was firstly amplified with the fungal ITS universal primers ITS1f/ITS4
(White et al., 1990; Gardes and Bruns, 1993), then the second PCR round was performed using T.
borchii and T. dryophilum species-specific primer pairs TboI-TboII and TdryI-TdryII (Amicucci et
al., 1998). The PCRs were carried out using a SimpliAmp thermal cycler (ThermoFisher, USA) and
PCR products were run on 1% agarose gel and visualized by staining with ethidium bromide.
    The presence of mycorrhizas was confirmed by detection of peloton structures using handmade
cross sections of orchid roots. These sections were gently cleared in hot 10% KOH (3 min at 70°C),
stained using a blue ink-vinegar solution (15 min at 70°C) (Vohník, 2020 modified) and observed under
an Eclipse TE 2000-E microscope (1000 X) (Nikon). The images were captured with a DXM1200F
digital camera (Nikon).

Results and discussion

In all the selected localities, several T. borchii and T. dryophilum ascomata were found during the
surveys as reported in the Table 2. However, only three T. dryophilum ascomata were found inside
the 10 m2 area around the fruiting point of T. borchii in Porto Corsini. That can be explained because
of T. borchii and T. dryophilum rarely share the same soil niches although they are found in the same
natural truffle grounds in Italy (Iotti et al., 2010; Zambonelli et al., 2012).



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    Orchid species found in the Appennine areas (Calderino and Guzzano) inside T. borchii production
patches were the green species Anacamptis morio (L.) R.M. Bateman, Pridgeon & M.W. Chas,
Orchis purpurea Huds., Cephalanthera longifolia (L.) Fritsch. and the achlorophyllous orchid
Limodorum abortivum (L.) Sw. In the truffle ground located in the littoral area in Porto Corsini only
the chlorophyllous Ophrys sphegodes Mill. was found in the T. borchii production patches. In our
root samples T. borchii was molecularly identified in 2 out of 6 samples of A. morio, in 3 out of 4
samples of C. longifolia and in 2 out of 6 samples of O. sphegodes (Table 2). Three orchids (OSP1,
OSP3 and OSP4) were found in the T. borchii patches were also a T. dryophilum ascoma was found;
in two of these (OSP1 and OSP3) T. borchii was identified inside the roots and T. dryophilum was
not detected by species specific primers. Considering the limited number of samples analyzed for T.
dryophilum orchid colonization we cannot exclude that also this species is able to colonize the orchid
roots. Further studies are necessary to verify this hypothesis.
    Morphological observations allowed to visualize the abundant presence of pelotons inside the
roots of the plants resulted infected with T. borchii (Fig. 1a,b) and of septate hyphae inside the
rhizoderma trichoblasts (Fig. 1c,d). Brown colored pelotons were also found which can be attributed
to the common Rhizoctonia-like mycorrhizas (Fig. 1e).


Table 2 - Results of the molecular analyses of the orchid species found in the 10 m2 area around T. borchii ascomata, listed
in the first column.
 T. borchii herbarium                                                                                   T. borchii
                               Orchid species           Sample                Locality
         number                                                                                       identification
            5263             Anacamptis morio           AMO1              Calderino (BO)                     -
            5264             Anacamptis morio           AMO2              Calderino (BO)                     -
            5265             Anacamptis morio           AMO3              Calderino (BO)                     -
            5266             Anacamptis morio           AMO4              Calderino (BO)                     x
            5267             Anacamptis morio           AMO5              Calderino (BO)                     -
            5268             Anacamptis morio           AMO6              Calderino (BO)                     x
            4613              Orchis purpurea            OPU1              Guzzano (BO)                      -
            4614              Orchis purpurea            OPU2              Guzzano (BO)                      -
            4615          Cephalanthera longifolia       CLO1              Guzzano (BO)                      x
            4617          Cephalanthera longifolia       CLO2              Guzzano (BO)                      x
            4018          Cephalanthera longifolia       CLO3              Guzzano (BO)                      x
            4019          Cephalanthera longifolia       CLO4              Guzzano (BO)                      -
            4021           Limodorum abortivum           LAB1              Guzzano (BO)                      -
            4027           Limodorum abortivum           LAB2              Guzzano (BO)                      -
          5285-1             Ophrys sphegodes           OSP1* Pineta di Porto Corsini (RA)                   x
          5285-2             Ophrys sphegodes            OSP2       Pineta di Porto Corsini (RA)             -
          5285-3             Ophrys sphegodes           OSP3* Pineta di Porto Corsini (RA)                   x
          5285-4             Ophrys sphegodes           OSP4* Pineta di Porto Corsini (RA)                   -
          5286-1             Ophrys sphegodes            OSP5       Pineta di Porto Corsini (RA)             -
          5287-2             Ophrys sphegodes            OSP6       Pineta di Porto Corsini (RA)             -
*Orchid samples (OSP1, OSP3 and OSP4) sampled in areas where a T. dryophilum ascoma was also found (herbarium
numbers 5283, 5284 and 5287 respectively).




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   The association between Tuber spp. (in particular T. maculatum, T. aestivum, Tuber excavatum
Vittad.) and orchids of the genus Cephalanthera has been reported in several studies (Julou et al.,
2005; Ouanphanivanh, 2008; Illyés et al., 2010) but T. borchii was found only able to associate to E.
atrorubens till now (Tešitelová et al., 2012). Moreover, this is the first study where doubtless a Tuber
spp. was found able to colonize the green orchids of the genera Anacamptis and Ophrys.
   The results obtained in this work are an important point to improve our knowledge on the range
of ECM fungi colonizing the orchids. The mycorrhizal association of orchids with ECM fungi has
revealed to be more common than we think and ECM fungi can adapt to a larger range of plant
hosts. This ability could have an important ecological effect; in fact, it may link different autotrophic
and mixotrophic organisms and allows the transport of nutrients through trees, mushrooms and
orchids (McKendrick et al., 2000; Julou et al., 2005). Many orchids are characterized by vegetative
dormancy, as adaptive strategy to enhance their long-term surviving possibilities against harsh years
(Shefferson et al., 2014). Thus, the mycelium may assist the orchids during their dormancy after a
stress, feeding them with nutrients those come from itself or other host plants. Moreover, Shefferson
et al. (2005) showed that C. longifolia can resist effectively to a shading treatment, with a high
survival rate and with less dormancy despite of other orchids. That could be related to its fungal
symbionts which provides nutrients during shading period. The carbon exchange between ECM fungi
and green orchids and the enhancement of the symbiotic partners biodiversity, may lead orchids
through a new evolutionary step, towards a fully mycoheterotrophic lifestyle and a total dependence
on fungal carbon (Julou et al., 2005).




            Fig. 1 - Pelotons colored with blue ink inside the roots of the plants resulted infected with T.
            borchii (a and b), septate hyphae inside the trichoblasts (c and d), brown pelotons formed by
            Rhizoctonia sp. (e).

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   However, the nature and physiological significance of ECM fungi present within rhizoctonia-
associated orchids is still not clear and need further investigation (Jacquemyn et al., 2017). In this
contest the association of orchids with Tuber species and particularly with T. borchii are especially
intriguing considering their common occurrence in the truffle production areas. Some authors
speculated that orchids and other herbaceous species could be involved in Tuber spp. life cycle
acting like reservoir of the paternal genotype (Qin and Feng, 2022). Moreover, the relation between
truffles and orchids may improve the surviving possibilities of both the organisms providing a protect
environment where the fungal mycelium can shelter during the hostile season.

Acknowledgements
The authors would like to thank the ‘Raggruppamento Carabinieri Biodiversità, Nucleo Tutela della
Biodiversità’ of Casalborsetti and Marina di Ravenna for giving as the permit to collect the orchids
and for providing very useful information and assistance in the field. We also thank the association
G.I.R.O.S for he help in orchids identificationa and Marco Morara, Guido Cicogni, Luigi Pelliconi,
Silvano Baraghini and their dogs for finding the truffles.

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