Mycorrhiza DOI 10.1007/s00572-014-0564-9
SHORT NOTE
Characterization of Tuber borchii and Arbutus unedo mycorrhizas Enrico Lancellotti & Mirco Iotti & Alessandra Zambonelli & Antonio Franceschini
Received: 15 October 2013 / Accepted: 30 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies. Keywords Arbutoid mycorrhizas . Bianchetto truffle . Mediterranean maquis . Truffle culture . Morphotyping
Introduction Arbutus unedo L. is widespread in the Mediterranean and Macaronesian area, with some Atlantic locations in France and Ireland (Villar 1993). Strawberry tree is well known in Mediterranean countries for the production of a highly valued monofloral bitter honey (Floris et al. 2007) and for its fruits that can be consumed in the field or used to make jams (Molina et al. 2011). Finally, this species is of great interest E. Lancellotti (*) : A. Franceschini Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy e-mail: [email protected] M. Iotti : A. Zambonelli Dipartimento di Scienze Agrarie, Università degli Studi di Bologna, via Fanin 46, 40127 Bologna, Italy
in gardening and landscaping (Navarro et al. 2007). A. unedo forms ectendo-type mycorrhizas, referred to as arbutoid, which are characterized by a fungal mantle with emanating elements (hyphae and/or cystidia), a well-developed Hartig net and an extensive intracellular development of hyphal coils in the epidermal root cells (Smith and Read 2008). As well as members of the genus Arbutus (Massicotte et al. 1993; Münzenberger et al. 1992), arbutoid mycorrhizas have also been described in Arctostaphylos (Read 1982; Muhlmann and Gobl 2006) and in the Pyrolae tribe (Robertson and Robertson 1985; Vincenot et al 2008), all taxa belonging to the Ericales. Some authors (Molina and Trappe 1982; Brundrett 2004; Imhof 2009) proposed arbutoid mycorrhizas as a type of ectomycorrhiza since the unique morpho-anatomical difference between these symbiotic structures concerns intracellular penetration, a feature that can be driven by the host plant (Brundrett 2004). Moreover, numerous fungal species identified in arbutoid mycorrhizas are ectomycorrhizal (Zak 1974; Molina and Trappe 1982; Krpata et al. 2007; Richard et al. 2009; Kennedy et al. 2012). Fungi that form arbutoid mycorrhizas belong mainly to the Basidiomycetes (Smith and Read 2008), but some Ascomycetes were also found associated with members of the Arbutoideae tribe (Zak 1974; Krpata et al. 2007; Kennedy et al. 2012). Molina and Trappe (1982) showed that fungi capable of forming arbutoid mycorrhizas were “host generalist” fungi; however, recent studies report that “host specialist” fungi, such as Lactarius deterrimus or Suillus plorans, were also linked with arbutoid plants (Muhlmann and Gobl 2006; Krpata et al. 2007). Tuber borchii Vittad., commonly called Bianchetto truffle, is a mycorrhizal Ascomycete belonging to the order Pezizales, that produces edible truffles. Its economic value is not as high as that of other edible truffles, like Tuber melanosporum and Tuber magnatum, but it is becoming increasingly renowned on the market (Hall et al. 2007). Its importance is due to both the gastronomic quality of its fruiting bodies as well as its
Mycorrhiza
ecological properties. T. borchii has a wide distribution in Europe, being found from southern Finland to Sicily and from Ireland to Hungary and Poland (Hall et al. 2007). Given its adaptability to different environmental conditions (Gardin 2005; Lancellotti and Franceschini 2013), it is considered the Tuber species with the widest ecological magnitude and it can be used to establish truffle plantations in environments unsuitable for other Tuber species, thus expanding the areas where it is possible to cultivate truffles (Zambonelli et al. 2002). In this work, we provide the evidence for symbiosis between T. borchii and A. unedo and describe their arbutoid mycorrhizas, both established in natural conditions and synthesized in the greenhouse.
Materials and methods Study area and sample processing The study area is a 50-year-old Pinus pinea L. wood (500 ha) located on the northwestern coast of Sardinia (Italy) (30 m a.s.l., UTM: 433741 E, 4503377 N). The shrub layer is dominated by holm oak (Quercus ilex L.), rock rose (Cistus creticus L.), narrow leaf phillirea (Phillyrea angustifolia L.), fan palm (Chamaerops umilis L.), prickly juniper (Juniperus oxicedrus L.), and strawberry tree (A. unedo L.), “the only species able to form arbutoid mycorrhizas present in the study area”. The soil is an Arenosol (FAO 1998) originating from a recent aeolian deposit of sand. The study area is characterized by a typical Mediterranean climate with cool wet winters and hot dry summers. Mean annual rainfall is from 400 to 500 mm (http://www.sar.sardegna.it). In February 2013, ascomata of T. borchii were harvested, with a trained dog, close to A. unedo shrubs and root samples were collected directly under each truffle found. Roots were carefully washed in sterile water and then examined under a dissecting stereomicroscope (×4–40) to identify the colonized tips with morpho-anatomical characters similar to those of T. borchii ectomycorrhizas (Giomaro et al. 2000). In particular, three mycorrhizal systems showing yellowish ochre and short spiny tips with a roundish to epidermoid pseudoparenchymatous mantle were isolated and maintained in FAA (formaldehyde; 70 % ethanol; acetic acid; 5:90:5) at 4 °C for morphological analysis. A few colonized tips representative of each ramified system were excised and stored in sterile water at −80 °C for molecular characterization. Molecular characterization Molecular characterization of a tip from the three different mycorrhizas was carried out by applying direct PCR approach as described by Iotti and Zambonelli (2006). The ITS1-5.8SITS2 rDNA region was amplified in a 50-μl volume reaction using the primer pair ITS1F-ITS4 (White et al. 1990; Gardes
and Bruns 1993) and a T gradient thermal cycler (Biometra). PCR reactions contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM for each dNTP, 300 nM for each primer, 30 μg of bovine serum albumin, and 1.5 U of Taq DNA polymerase (TaKaRa). Thermal cycler conditions were the following: 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an initial denaturation at 94 °C for 8 min and a final extension at 72 °C for 10 min. Amplicons were electrophoresed in a 1 % agarose gel and visualized by staining with ethidium bromide in a GeneGenius Imaging System (Syngene). Purification was carried out with the NucleoSpin Extract II kit (Macherey-Nagel) and sequencing was performed using the Abi Prism 3700 DNA Analyzer (Applied Biosystem, Foster City, CA) with Big Dye Terminator v3.1 chemistry. The ITS sequences obtained were compared with those present in GenBank database (http://www.ncbi.nlm.nih. gov/BLAST/). Only one representative of three obtained sequences was deposited in GenBank database with the accession number KF529975. Morphological characterization Morphology and anatomy of arbutoid mycorrhizas collected in the study area were described by a set of characteristics after Agerer (1987–2008). Morphology of the three ramified systems and their unramified ends was recorded under a Stemi SV 11 dissecting microscope (×40) (Zeiss); images were captured using a Moticam 2300 digital camera and measurements taken using Motic Image 2.0 plus software. The anatomical structures of the mantle and external elements (hyphae and cystidia) of three unramified ends of each ramified system were examined in plan view and longitudinal sections under an ECLIPSE TE 2000-E microscope (×1,000) (Nikon) equipped with differential interference contrast optics (Normanski) and video enhancement. Digital photos and measurements were taken using an Nis-Elements AR (v 3.10) software (Zeiss) from images captured with a DXM1200F digital camera (Nikon). Cross sections (8 to 10-μm thickness) were obtained by embedding unramified ends in Technovit 7100 resin (Kulzer) according to the manufacturer’s recommendations and then cutting them with a Leitz 1512 microtome. Synthesis of T. borchii+A. unedo mycorrhizas T. borchii mycorrhizas were synthesized under greenhouse conditions using mycelial inoculation techniques and A. unedo as host plant. Mycelium of T. borchii (strain 56SS) was isolated in March 2005 from an ascoma collected in a Q. ilex stand in Sardinia. It has been maintained on potato dextrose agar half strength (20 g/l) at 22 °C and subcultured every 2 months. Seeds of A. unedo were collected in autumn 2012, sterilized in a 1 % sodium hypochlorite solution for
Mycorrhiza
10 min, rinsed under tap water, and kept at 4 °C in moist sterile sand until use. They were placed to germinate in a peat sand mixture (1:9) under greenhouse conditions, and the developing root systems were washed and pruned before inoculation. Inoculum was prepared by culturing mycelium in flasks containing 100 ml of modified woody plant medium (mWPM) (Iotti et al. 2005) at 22 °C, in the dark, for 40 days. Mycelia were then transferred to 200 ml of a sterile peat: vermiculite mix (1:9) imbibed by 60 ml of mWPM. After further 40 days of incubation, the colonized substrate was wrapped in a sterile gauze, rinsed and squeezed five times under tap water, and used to inoculate five 1-month-old seedlings. Inoculum was mixed into a sandy soil (pH 7.5) sterilized twice at 120 °C for 1 h at a proportion of 1:5. Inoculated seedlings were grown in plastic pots (6 cm in diameter, 15-cm high), kept in a greenhouse under natural daylight conditions, and watered weekly with 200 ml of tap water. Completely formed mycorrhizas were collected from each seedling 3 months after inoculation.
Results Molecular characterization Molecular analysis of A. unedo mycorhizas from the field generated 605-bp sequences with a similarity of 100 %. Blast search against GenBank database showed a full identity with sequences of T. borchii belonging to the lineage 1 according to Bonuso et al. (2009). Morphological characterization Morphology Mycorrhizal system, 2.63-mm long and 3.67mm wide, carried one, three, or six mycorrhizas (Fig. 1a, b). Mycorrhiza were orange in color, 1.1-mm long, and 1.6-mm wide; ramifications were cruciform with three unramified ends which were 0.65-mm long with a diameter of 0.31 mm. The angle between adjacent unramified ends was 65°; they were orange in color with whitish ends; the surface was shiny with short spines more frequent in distal parts, and cortical cells were visible. Anatomy Mantle was pseudoparenchymatous with epidermoid cells (Fig. 1c, d). Mean area of mantle cells was 113.67±4.37 μm, and mean perimeter was 55.96±1.27 μm (n=50). Cystidia were bristle like, 82.15±3.75-μm long, and 3.71±0.12-μm (n=15) wide at the base (Fig. 1g). The longitudinal section showed mantle composed of four–six hyphal layers, 18.67-μm thick (Fig. 1d). Hartig net involved only the epidermal cells which were also colonized by hyphal complex (Fig. 1e, f).
Synthesis of T. borchii+A. unedo mycorrhizas Three months after inoculation, all five inoculated A. unedo seedlings were colonized by T. borchii (strain 56SS) and, on average, 50 % of fine roots were mycorrhizal. Arbutoid mycorrhizas obtained in greenhouse conditions showed the same morpho-anatomical characteristics as those collected in the field.
Discussion The present study is the first report to describe arbutoid mycorrhizas generated from T. borchii either in natural or in controlled conditions. Symbiosis between species of Tuber and arbutoid plants was found using the molecular method by Kennedy et al. (2012). However, according to our knowledge, arbutoid mycorrhizas formed by Tuber genus have not yet been described and have never been considered as a new perspective for truffle cultivation. T. borchii and A. unedo mycorrhizas are typical arbutoid mycorrhizas which, however, show many characteristics typical of ectomycorrhizas formed by T. borchii (Zambonelli and Branzanti 1984; Zambonelli et al. 1993, 1995). Orange color, surface with short spines, mantle with epidermoid cells, and awl-shaped cystidia are typical and constant characteristics of mycorrhizas of T. borchii. Biometric characteristics, for example, mantle cell area or cystidia length, were also included in the variability observed on ectomycorrhizas of this fungal taxon (Giomaro et al. 2000). The main differences were the cruciform branching of mycorrhizas and intracellular colonization of host cells, which are characteristics of arbutoid mycorrhizas (Molina and Trappe 1982). Moreover, the ability of T. borchii to differentially interact with the plant forming arbutoid mycorrhizas, ectomycorrhizas or orchid mycorrhizas (GenBank accession number GU327399 deposited by Tešitelová et al. 2012) may be an additional opportunity to increase our knowledge about the molecular signals involved in the formation of symbiosis. In the framework of truffle cultivation, the ability of T. borchii to establish symbiosis with A. unedo in natural conditions and to easily colonize its seedlings in the greenhouse increases the number of host plant/fungus combinations that can be used in truffle cultivation. A. unedo/T. borchii plantations can be considered multipurpose forestations with entomologic (honey), agronomic (fruit), and mycological (truffle) production. This aspect may make this culture economically sustainable and represents a promising alternative for marginal lands and particularly for semiarid Mediterranean areas unsuitable for the cultivation of the most precious truffles (T. melanosporum and T. magnatum) (Zambonelli et al. 2014). Furthermore, symbiosis between T. borchii and A. unedo also has significant ecological implications. It is known that
Mycorrhiza Fig. 1 a–g Morphology and anatomy of T. borchii–A. unedo arbutoid mycorrhizas. a Branched mycorrhizal rootlet (bar=500 μm), b typical cruciform branching (bar=200 μm), c outer layer of the fungal mantle in plan view (bar=10 μm), d fungal mantle and colonized epidermal cells in cross section (bar=10 μm), e early stage of root cell colonization (bar=10 μm), f root cell completely colonized by T. borchii mycelium (bar=10 μm), and g cystidia of T. borchii (bar=10 μm). Arrows indicate the point of hyphal penetration into epidermal cells
a
b
c
d
e
f
g
A. unedo facilitates Q. ilex seedling establishment by providing compatible ectomycorrhizal fungal symbionts in seral successional stages of Mediterranean forest (Richard et al. 2009). In fact, the availability of compatible fungal inoculum in soil locally influences the ability of tree seedlings to establish and contributes to plant community dynamics (Horton et al. 1999; Azcon-Aguilar et al. 2003; Dickie et al. 2004; Nara 2006; Selosse and Duplessis 2006). T. borchii establishes symbiosis with a wide range of host species, including orchids (Tešitelová et al. 2012). It typically forms ectomycorrhizas
with species of Pinaceae and Fagaceae, but it has also been found associated with Cedrus spp. and Populus spp. (Zambonelli et al. 2002; Hall et al. 2007). In littoral areas of Italy, it was commonly found associated with P. pinea, Pinus pinaster, and Q. ilex (Iotti et al. 2010). Considering the low host specificity of T. borchii, A. unedo/T. borchii plantations may also benefit other plant species by providing nutrient and water resources via access to extensive mycelial networks (Van der Heijden and Horton 2009; Simard et al. 2012). Consequently, the A. unedo/T. borchii plants could help
Mycorrhiza
forestation programs in Mediterranean areas by linking the economically advantageous production of truffle and fruit to a positive influence on seedling survival.
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Massicotte HB, Melville LH, Molina R, Peterson RL (1993) Structure and histochemistry of mycorrhizae synthesized between Arbutus menziesii (Ericaceae) and two basidiomycetes, Pisolithus tinctorius (Pisolithaceae) and Piloderma bicolor (Corticiaceae). Mycorrhiza 3: 1–11 Molina R, Trappe JM (1982) Lack of mycorrhizal specificity in the ericaceous hosts Arbutus menziessi and Arctostaphylos uva-ursi. New Phytol 90:495–509 Molina M, Pardo-De-Santayana M, Aceituno L, Morales R, Tardio J (2011) Fruit production of strawberry tree (Arbutus unedo L.) in two Spanish forests. Forestry 84:419–429 Muhlmann O, Gobl F (2006) Mycorrhiza of the host-specific Lactarius deterrimus on the roots of Picea abies and Arctostaphylos uva-ursi. Mycorrhiza 16:245–250 Münzenberger B, Kottke I, Oberwinkler F (1992) Ultrastructural investigations of Arbutus unedo-Laccaria amethystea mycorrhiza synthesized in vitro. Trees 7:40–47 Nara K (2006) Pioneer dwarf willow may facilitate tree succession by providing late colonizers with compatible ectomycorrhizal fungi in a primary successional volcanic desert. New Phytol 171:187–198 Navarro A, Sánchez-Blanco MJ, Bañón S (2007) Influence of paclobutrazol on water consumption and plant performance of Arbutus unedo seedlings. Sci Hortic 111:133–139 Read DJ (1982) The biology of mycorrhiza in the Ericales. Can J Bot 61: 985–1004 Richard F, Selosse M-A, Gardes M (2009) Facilitated establishment of Quercus ilex in shrub-dominated communities within a Mediterranean ecosystem: do mycorrhizal partners matter? FEMS Microbiol Ecol 68:14–24 Robertson DC, Robertson JA (1985) Ultrastructural aspects of Pyrola mycorrhizae. Can J Bot 63:1089–1098 Selosse M-A, Duplessis S (2006) More complexity in the mycorrhizas world. New Phytol 172:600–604 Simard SW, Beiler KJ, Bingham MA, Deslippe JR, Philip LJ, Teste FP (2012) Mycorrhizal networks: mechanisms, ecology and modelling. Fung Biol Rev 26:39–60 Smith SE, Read DJ (2008) Mycorrhizal symbiosis, 3rd edn. Academic Press, New York Tešitelová T, Tešitel J, Jersáková J, Ríhová G, Selosse M-A (2012) Symbiotic germination capability of four Epipactis species (Orchidaceae) is broader than expected from adult ecology. Am J Bot 99:1020–1032 Van der Heijden MGA, Horton TR (2009) Socialism in soil? The importance of mycorrhizal fungal networks for facilitation in natural ecosystems. J Ecol 97:1139–1150 Villar L (1993) Arbutus L. In: Castroviejo S, Aedo C, Gómez C, Laínz M, Montserrat P, Morales R, Muñoz F, Nieto G, Rico E, Talavera S, Villar L (eds) Flora ibérica, vol IV. CSIC, Madrid, pp 514–516 Vincenot L, Tedersoo L, Richard F, Horcine H, Kõljalg U, Selosse M-A (2008) Fungal associates of Pyrola rotundifolia, a mixotrophic Ericaceae, from two Estonian boreal forests. Mycorrhiza 19:15–25 White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, San Diego, pp 315–321 Zak (1974) Ectendomycorrhiza of Pacific madrone (Arbutus menziesii). Trans Br Mycol Soc 62:202–204 Zambonelli A, Branzanti MB (1984) Prove di micorrizazione del nocciolo con Tuber aestivum e Tuber albidum. Micol Ital 13:47–52 Zambonelli A, Salomoni S, Pisi A (1993) Caratterizzazione anatomomorphologica delle micorrize di Tuber spp. su Quercus pubescens Willd. Micol Ital 22:73–90
Mycorrhiza Zambonelli A, Salomoni S, Pisi A (1995) Caratterizzazione anatomomorfologica delle micorrize di Tuber borchii, Tuber aestivum, Tuber mesentericum, Tuber brumale, Tuber melanosporum su Pinus pinea. Micol Ital 24:119–137 Zambonelli A, Iotti M, Giomaro G, Hall I, Stocchi V (2002) In: Hall I, Yun W, Danell E, Zambonelli A (eds) T. borchii cultivation: an interesting perspective. Edible mycorrhizal mushrooms and their cultivation. Proceedings of the Second International Conference on
Edible Mycorrhizal Mushrooms, CD ROM, 3–6 July 2001, Christchurch, New Zealand. New Zealand Institute for Crop and Food Research Limited, Christchurch Zambonelli A, Donnini D, Rana GL, Fascetti S, Benucci GMN, Iotti M, Morte A, Khabar L, Bawadekji A, Piattoni F, Compagno R, Venturella G (2014) Hypogeous fungi in Mediterranean maquis, arid and semi-arid forests. Plant Biosystem. doi:10.1080/ 11263504.2013.877537
SHORT NOTE
Characterization of Tuber borchii and Arbutus unedo mycorrhizas Enrico Lancellotti & Mirco Iotti & Alessandra Zambonelli & Antonio Franceschini
Received: 15 October 2013 / Accepted: 30 January 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract For the first time, arbutoid mycorrhizas established between Tuber borchii and Arbutus unedo were described. Analyzed mycorrhizas were from one T. borchii natural truffle ground, dominated by Pinus pinea, as well as synthesized in greenhouse conditions. A. unedo mycorrhizas presented some typical characteristics of ectomycorrhizas of T. borchii. However, as in arbutoid mycorrhizas, ramification was cruciform and intracellular colonization in epidermal cells was present. The ability of T. borchii to form ectomycorrhizas with A. unedo opens up the possibility to also use this fruit plant for truffle cultivation. This represents an important economic opportunity in Mediterranean areas by combining both the cultivation of precious truffles and the production of edible fruits which are used fresh or in food delicacies. Keywords Arbutoid mycorrhizas . Bianchetto truffle . Mediterranean maquis . Truffle culture . Morphotyping
Introduction Arbutus unedo L. is widespread in the Mediterranean and Macaronesian area, with some Atlantic locations in France and Ireland (Villar 1993). Strawberry tree is well known in Mediterranean countries for the production of a highly valued monofloral bitter honey (Floris et al. 2007) and for its fruits that can be consumed in the field or used to make jams (Molina et al. 2011). Finally, this species is of great interest E. Lancellotti (*) : A. Franceschini Dipartimento di Agraria, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy e-mail: [email protected] M. Iotti : A. Zambonelli Dipartimento di Scienze Agrarie, Università degli Studi di Bologna, via Fanin 46, 40127 Bologna, Italy
in gardening and landscaping (Navarro et al. 2007). A. unedo forms ectendo-type mycorrhizas, referred to as arbutoid, which are characterized by a fungal mantle with emanating elements (hyphae and/or cystidia), a well-developed Hartig net and an extensive intracellular development of hyphal coils in the epidermal root cells (Smith and Read 2008). As well as members of the genus Arbutus (Massicotte et al. 1993; Münzenberger et al. 1992), arbutoid mycorrhizas have also been described in Arctostaphylos (Read 1982; Muhlmann and Gobl 2006) and in the Pyrolae tribe (Robertson and Robertson 1985; Vincenot et al 2008), all taxa belonging to the Ericales. Some authors (Molina and Trappe 1982; Brundrett 2004; Imhof 2009) proposed arbutoid mycorrhizas as a type of ectomycorrhiza since the unique morpho-anatomical difference between these symbiotic structures concerns intracellular penetration, a feature that can be driven by the host plant (Brundrett 2004). Moreover, numerous fungal species identified in arbutoid mycorrhizas are ectomycorrhizal (Zak 1974; Molina and Trappe 1982; Krpata et al. 2007; Richard et al. 2009; Kennedy et al. 2012). Fungi that form arbutoid mycorrhizas belong mainly to the Basidiomycetes (Smith and Read 2008), but some Ascomycetes were also found associated with members of the Arbutoideae tribe (Zak 1974; Krpata et al. 2007; Kennedy et al. 2012). Molina and Trappe (1982) showed that fungi capable of forming arbutoid mycorrhizas were “host generalist” fungi; however, recent studies report that “host specialist” fungi, such as Lactarius deterrimus or Suillus plorans, were also linked with arbutoid plants (Muhlmann and Gobl 2006; Krpata et al. 2007). Tuber borchii Vittad., commonly called Bianchetto truffle, is a mycorrhizal Ascomycete belonging to the order Pezizales, that produces edible truffles. Its economic value is not as high as that of other edible truffles, like Tuber melanosporum and Tuber magnatum, but it is becoming increasingly renowned on the market (Hall et al. 2007). Its importance is due to both the gastronomic quality of its fruiting bodies as well as its
Mycorrhiza
ecological properties. T. borchii has a wide distribution in Europe, being found from southern Finland to Sicily and from Ireland to Hungary and Poland (Hall et al. 2007). Given its adaptability to different environmental conditions (Gardin 2005; Lancellotti and Franceschini 2013), it is considered the Tuber species with the widest ecological magnitude and it can be used to establish truffle plantations in environments unsuitable for other Tuber species, thus expanding the areas where it is possible to cultivate truffles (Zambonelli et al. 2002). In this work, we provide the evidence for symbiosis between T. borchii and A. unedo and describe their arbutoid mycorrhizas, both established in natural conditions and synthesized in the greenhouse.
Materials and methods Study area and sample processing The study area is a 50-year-old Pinus pinea L. wood (500 ha) located on the northwestern coast of Sardinia (Italy) (30 m a.s.l., UTM: 433741 E, 4503377 N). The shrub layer is dominated by holm oak (Quercus ilex L.), rock rose (Cistus creticus L.), narrow leaf phillirea (Phillyrea angustifolia L.), fan palm (Chamaerops umilis L.), prickly juniper (Juniperus oxicedrus L.), and strawberry tree (A. unedo L.), “the only species able to form arbutoid mycorrhizas present in the study area”. The soil is an Arenosol (FAO 1998) originating from a recent aeolian deposit of sand. The study area is characterized by a typical Mediterranean climate with cool wet winters and hot dry summers. Mean annual rainfall is from 400 to 500 mm (http://www.sar.sardegna.it). In February 2013, ascomata of T. borchii were harvested, with a trained dog, close to A. unedo shrubs and root samples were collected directly under each truffle found. Roots were carefully washed in sterile water and then examined under a dissecting stereomicroscope (×4–40) to identify the colonized tips with morpho-anatomical characters similar to those of T. borchii ectomycorrhizas (Giomaro et al. 2000). In particular, three mycorrhizal systems showing yellowish ochre and short spiny tips with a roundish to epidermoid pseudoparenchymatous mantle were isolated and maintained in FAA (formaldehyde; 70 % ethanol; acetic acid; 5:90:5) at 4 °C for morphological analysis. A few colonized tips representative of each ramified system were excised and stored in sterile water at −80 °C for molecular characterization. Molecular characterization Molecular characterization of a tip from the three different mycorrhizas was carried out by applying direct PCR approach as described by Iotti and Zambonelli (2006). The ITS1-5.8SITS2 rDNA region was amplified in a 50-μl volume reaction using the primer pair ITS1F-ITS4 (White et al. 1990; Gardes
and Bruns 1993) and a T gradient thermal cycler (Biometra). PCR reactions contained 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 mM for each dNTP, 300 nM for each primer, 30 μg of bovine serum albumin, and 1.5 U of Taq DNA polymerase (TaKaRa). Thermal cycler conditions were the following: 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with an initial denaturation at 94 °C for 8 min and a final extension at 72 °C for 10 min. Amplicons were electrophoresed in a 1 % agarose gel and visualized by staining with ethidium bromide in a GeneGenius Imaging System (Syngene). Purification was carried out with the NucleoSpin Extract II kit (Macherey-Nagel) and sequencing was performed using the Abi Prism 3700 DNA Analyzer (Applied Biosystem, Foster City, CA) with Big Dye Terminator v3.1 chemistry. The ITS sequences obtained were compared with those present in GenBank database (http://www.ncbi.nlm.nih. gov/BLAST/). Only one representative of three obtained sequences was deposited in GenBank database with the accession number KF529975. Morphological characterization Morphology and anatomy of arbutoid mycorrhizas collected in the study area were described by a set of characteristics after Agerer (1987–2008). Morphology of the three ramified systems and their unramified ends was recorded under a Stemi SV 11 dissecting microscope (×40) (Zeiss); images were captured using a Moticam 2300 digital camera and measurements taken using Motic Image 2.0 plus software. The anatomical structures of the mantle and external elements (hyphae and cystidia) of three unramified ends of each ramified system were examined in plan view and longitudinal sections under an ECLIPSE TE 2000-E microscope (×1,000) (Nikon) equipped with differential interference contrast optics (Normanski) and video enhancement. Digital photos and measurements were taken using an Nis-Elements AR (v 3.10) software (Zeiss) from images captured with a DXM1200F digital camera (Nikon). Cross sections (8 to 10-μm thickness) were obtained by embedding unramified ends in Technovit 7100 resin (Kulzer) according to the manufacturer’s recommendations and then cutting them with a Leitz 1512 microtome. Synthesis of T. borchii+A. unedo mycorrhizas T. borchii mycorrhizas were synthesized under greenhouse conditions using mycelial inoculation techniques and A. unedo as host plant. Mycelium of T. borchii (strain 56SS) was isolated in March 2005 from an ascoma collected in a Q. ilex stand in Sardinia. It has been maintained on potato dextrose agar half strength (20 g/l) at 22 °C and subcultured every 2 months. Seeds of A. unedo were collected in autumn 2012, sterilized in a 1 % sodium hypochlorite solution for
Mycorrhiza
10 min, rinsed under tap water, and kept at 4 °C in moist sterile sand until use. They were placed to germinate in a peat sand mixture (1:9) under greenhouse conditions, and the developing root systems were washed and pruned before inoculation. Inoculum was prepared by culturing mycelium in flasks containing 100 ml of modified woody plant medium (mWPM) (Iotti et al. 2005) at 22 °C, in the dark, for 40 days. Mycelia were then transferred to 200 ml of a sterile peat: vermiculite mix (1:9) imbibed by 60 ml of mWPM. After further 40 days of incubation, the colonized substrate was wrapped in a sterile gauze, rinsed and squeezed five times under tap water, and used to inoculate five 1-month-old seedlings. Inoculum was mixed into a sandy soil (pH 7.5) sterilized twice at 120 °C for 1 h at a proportion of 1:5. Inoculated seedlings were grown in plastic pots (6 cm in diameter, 15-cm high), kept in a greenhouse under natural daylight conditions, and watered weekly with 200 ml of tap water. Completely formed mycorrhizas were collected from each seedling 3 months after inoculation.
Results Molecular characterization Molecular analysis of A. unedo mycorhizas from the field generated 605-bp sequences with a similarity of 100 %. Blast search against GenBank database showed a full identity with sequences of T. borchii belonging to the lineage 1 according to Bonuso et al. (2009). Morphological characterization Morphology Mycorrhizal system, 2.63-mm long and 3.67mm wide, carried one, three, or six mycorrhizas (Fig. 1a, b). Mycorrhiza were orange in color, 1.1-mm long, and 1.6-mm wide; ramifications were cruciform with three unramified ends which were 0.65-mm long with a diameter of 0.31 mm. The angle between adjacent unramified ends was 65°; they were orange in color with whitish ends; the surface was shiny with short spines more frequent in distal parts, and cortical cells were visible. Anatomy Mantle was pseudoparenchymatous with epidermoid cells (Fig. 1c, d). Mean area of mantle cells was 113.67±4.37 μm, and mean perimeter was 55.96±1.27 μm (n=50). Cystidia were bristle like, 82.15±3.75-μm long, and 3.71±0.12-μm (n=15) wide at the base (Fig. 1g). The longitudinal section showed mantle composed of four–six hyphal layers, 18.67-μm thick (Fig. 1d). Hartig net involved only the epidermal cells which were also colonized by hyphal complex (Fig. 1e, f).
Synthesis of T. borchii+A. unedo mycorrhizas Three months after inoculation, all five inoculated A. unedo seedlings were colonized by T. borchii (strain 56SS) and, on average, 50 % of fine roots were mycorrhizal. Arbutoid mycorrhizas obtained in greenhouse conditions showed the same morpho-anatomical characteristics as those collected in the field.
Discussion The present study is the first report to describe arbutoid mycorrhizas generated from T. borchii either in natural or in controlled conditions. Symbiosis between species of Tuber and arbutoid plants was found using the molecular method by Kennedy et al. (2012). However, according to our knowledge, arbutoid mycorrhizas formed by Tuber genus have not yet been described and have never been considered as a new perspective for truffle cultivation. T. borchii and A. unedo mycorrhizas are typical arbutoid mycorrhizas which, however, show many characteristics typical of ectomycorrhizas formed by T. borchii (Zambonelli and Branzanti 1984; Zambonelli et al. 1993, 1995). Orange color, surface with short spines, mantle with epidermoid cells, and awl-shaped cystidia are typical and constant characteristics of mycorrhizas of T. borchii. Biometric characteristics, for example, mantle cell area or cystidia length, were also included in the variability observed on ectomycorrhizas of this fungal taxon (Giomaro et al. 2000). The main differences were the cruciform branching of mycorrhizas and intracellular colonization of host cells, which are characteristics of arbutoid mycorrhizas (Molina and Trappe 1982). Moreover, the ability of T. borchii to differentially interact with the plant forming arbutoid mycorrhizas, ectomycorrhizas or orchid mycorrhizas (GenBank accession number GU327399 deposited by Tešitelová et al. 2012) may be an additional opportunity to increase our knowledge about the molecular signals involved in the formation of symbiosis. In the framework of truffle cultivation, the ability of T. borchii to establish symbiosis with A. unedo in natural conditions and to easily colonize its seedlings in the greenhouse increases the number of host plant/fungus combinations that can be used in truffle cultivation. A. unedo/T. borchii plantations can be considered multipurpose forestations with entomologic (honey), agronomic (fruit), and mycological (truffle) production. This aspect may make this culture economically sustainable and represents a promising alternative for marginal lands and particularly for semiarid Mediterranean areas unsuitable for the cultivation of the most precious truffles (T. melanosporum and T. magnatum) (Zambonelli et al. 2014). Furthermore, symbiosis between T. borchii and A. unedo also has significant ecological implications. It is known that
Mycorrhiza Fig. 1 a–g Morphology and anatomy of T. borchii–A. unedo arbutoid mycorrhizas. a Branched mycorrhizal rootlet (bar=500 μm), b typical cruciform branching (bar=200 μm), c outer layer of the fungal mantle in plan view (bar=10 μm), d fungal mantle and colonized epidermal cells in cross section (bar=10 μm), e early stage of root cell colonization (bar=10 μm), f root cell completely colonized by T. borchii mycelium (bar=10 μm), and g cystidia of T. borchii (bar=10 μm). Arrows indicate the point of hyphal penetration into epidermal cells
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A. unedo facilitates Q. ilex seedling establishment by providing compatible ectomycorrhizal fungal symbionts in seral successional stages of Mediterranean forest (Richard et al. 2009). In fact, the availability of compatible fungal inoculum in soil locally influences the ability of tree seedlings to establish and contributes to plant community dynamics (Horton et al. 1999; Azcon-Aguilar et al. 2003; Dickie et al. 2004; Nara 2006; Selosse and Duplessis 2006). T. borchii establishes symbiosis with a wide range of host species, including orchids (Tešitelová et al. 2012). It typically forms ectomycorrhizas
with species of Pinaceae and Fagaceae, but it has also been found associated with Cedrus spp. and Populus spp. (Zambonelli et al. 2002; Hall et al. 2007). In littoral areas of Italy, it was commonly found associated with P. pinea, Pinus pinaster, and Q. ilex (Iotti et al. 2010). Considering the low host specificity of T. borchii, A. unedo/T. borchii plantations may also benefit other plant species by providing nutrient and water resources via access to extensive mycelial networks (Van der Heijden and Horton 2009; Simard et al. 2012). Consequently, the A. unedo/T. borchii plants could help
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forestation programs in Mediterranean areas by linking the economically advantageous production of truffle and fruit to a positive influence on seedling survival.
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