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Molecular identification of Tuber magnatum ectomycorrhizae in the field
ARTICLE IN PRESS Microbiological Research 161 (2006) 59—64
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Molecular identification of Tuber magnatum ectomycorrhizae in the field Luana Bertinia, Ismaela Rossia, Alessandra Zambonellib, Antonella Amicuccia, Achille Sacchic, Matteo Cecchinid, Gianluigi Gregoric, Vilberto Stocchia, a
Istituto di Chimica Biologica Giorgio Fornaini, Universita ` degli Studi di Urbino, Carlo Bo, Via Saffi 2, 61029 Urbino (PU), Italy b Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita ` di Bologna, Via Fanin 46, 40127 Bologna, Italy c Centro Sperimentale di Tartuficoltura, Regione Marche, Via Macina 2, 61048 Sant’Angelo in Vado (PU), Italy d Istituto e Orto Botanico, Cattedra di Botanica, Universita ` degli Studi di Urbino, Via Bramante 28, 61029 Urbino (PU), Italy. Accepted 8 June 2005
KEYWORDS Tuber magnatum; Ectomycorrhizae; Molecular identification; ‘‘truffie `re’’
Summary Tuber ectomycorrhizae in a Tuber magnatum ‘‘truffie `re’’, located in Central Italy, were studied using molecular methods. Specifically, RFLP–ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the ‘‘truffie `re’’ under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively). & 2005 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +39 0722 305262; fax: +39 0722 320188.
E-mail address: [email protected] (V. Stocchi). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.06.003
ARTICLE IN PRESS 60
L. Bertini et al.
Introduction Truffles are the fruiting bodies of ascomycetous fungi belonging to the genus Tuber, which form ectomycorrhizae on the roots of angiosperms and gymnosperms. Some Tuber species, such as Tuber magnatum, produce edible fruiting bodies. T. magnatum is the most highly regarded truffle, with a market demand far exceeding the quantities that can be harvested from its natural wild habitats, primarily in Italy and Istria (Croatia) (Hall et al. 1998). Unlike Tuber melanosporum, Tuber aestivum and Tuber borchii, T. magnatum has not been successfully cultivated in truffle beds (Tibiletti and Zambonelli 1999). This is partly because it is difficult to obtain plants infected with T. magnatum that are uncontaminated by other species of white truffle, such as T. maculatum and T. borchii, or Sphaerosporella brunnea (Danielson 1984) and Pulvinula constellatio (Amicucci et al. 2001). T. maculatum, T. borchii and other Tuber species also seem to actively compete with T. magnatum when artificially infected plants are planted in ‘‘truffie `res’’ (Donnini et al. 2000). In order to improve T. magnatum cultivation and to identify the specific requirements of this fungus, many ecological studies have been carried out in natural ‘‘truffie `res’’ (Hall et al. 1998). However, very few studies have been conducted on the ectomycorrhizal communities of these productive areas. In all previous investigations mycorrhizae were identified using morphological methods,
Table 1.
which are imprecise and do not allow us to unequivocally distinguish T. magnatum mycorrhizae from those of T. maculatum, T. dryophilum and T. borchii (Gregori 2002). The present study uses molecular methods to investigate the ectomycorrhizal communities of different Tuber species in a productive T. magnatum ‘‘truffie `re’’.
Materials and methods Geographical location and ecological characteristics of the natural ‘‘truffie `re The ‘‘truffie `re’’ under investigation is located in Central Italy in the Marches region, in an area in the province of Pesaro-Urbino known as ‘‘Il Colle’’, situated 270 m a.s.l., at 431460 4300 latitude and 121450 2000 longitude. Covering about 6 ha, the ‘‘truffie `re’’ lies in a cool shady valley, exposed to the east. The climate is sub-Mediterranean and rainfall usually exceeds 900 mm per year mainly falling around the spring and autumn equinoxes with a high peak in the autumn and a second lower peak in the spring typical of Adriatic inland areas (Bocci 1982). The physical–chemical characteristics of the soil in the ‘‘truffie `re’’ are reported in Table 1. The ‘‘truffie `re’’ is almost completely covered by vegetation (about 95%), consisting of herbaceous (60%), shrub (18%) and arboreal species (22%). The T. magnatum host plants are oaks, poplars, willows
Physical–chemical characteristics of soil
Parameters
Average
Maximum values
Minimum values
Standard deviation
Sand (g/kg) Mud (g/kg) Clay (g/kg) pH (H2O) pH (KCl) Total carbonate (g/kg) Active calcium carbonate (g/kg) Organic matter (g/kg) Total nitrogen (g/kg) Phosphate (assimilable) (mg/kg) Potassium (exchangeable) (mg/kg) Magnesium (exchangeable) (mg/kg) Cation exchange capacity (meq/100 g) Iron (assimilable) (mg/kg) Manganese (assimilable) (mg/kg) Zinc (assimilable) (mg/kg) Copper (assimilable) (mg/kg)
354.0 470.7 175.3 8.0 7.6 201.5 45.4 27.4 1.6 4.0 271.6 392.7 27.4 22.8 19.4 2.7 2.5
505.0 589.0 298.0 8.5 8.1 280.0 101.0 46.7 2.5 8.0 520.0 504.0 34.8 41.5 26.6 4.6 3.5
134.0 380.0 105.0 7.6 7.3 160.0 24.0 15.7 1.1 1.0 160.0 321.0 21.1 10.4 11.1 1.9 1.5
116.0 77.7 63.8 0.2 0.2 2.8 24.8 11.6 0.5 2.2 106.1 69.0 4.7 9.1 6.1 0.9 0.7
ARTICLE IN PRESS Molecular identification of Tuber magnatum ectomycorrhizae in the field Table 2.
61
Representative plant species present in ‘‘truffie `re’’ under study
Arboreal species
Shrub species
Herbaceous vegetation
Ostrya carpinifolia Populus alba Populus nigra Populus tremula Quercus pubescens Salix alba Salix caprea
Clematis vitalba Cornus sanguinea Euonymus europaeus Hedera helix Prunus spinosa Rubus caesius Rubus ulmifolius
Brachypodium sylvaticum Equisetum arvense Equisetum telmateja Eupatorium cannabinum Euphorbia cyparissias Pteridium aquilinum Ranunculus acris Symphytum tuberosum Tamus communis
and hornbeam (Table 2), and the ‘‘truffie `re’’ has produced about 15 kg of truffles per hectare over the last 5 years (Gregori et al. 2002).
Sample collection The soil samples were collected using a 60 mm diameter soil corer at depths of 0–15 cm, 15–30 cm and 30–45 cm at three different times in 2001: May (2 May, 12 May), June–July (28 June, 5 July, 12 July) and September–October (7 September, 15 September, 16 October). A total of 90 soil samples were taken. Sampling was done only in the more productive areas in order to increase the probability of finding natural T. magnatum ectomycorrhizae. The soil samples were stored overnight at 4 1C and were separated from the soil using a 0.5 mm mesh sieve.
regions were amplified with the primers ITS1/ ITS4, ITS1/ITS2, ITS5/ITS6 (White et al. 1990; Bertini et al. 1999). The primers were synthesized by Sigma-Aldrich (Poole, UK). The RFLP (Restriction Fragment Length Polymorphism) analyses with Taq I and Rsa I enzymes (Takara Biomedicals) of the amplified ITS 5–6 region were performed on unpurified PCR products (Bertini et al. 1998). The amplified ITS 5–6 region was first purified with the QIAquick PCR purification kit or with the gel extraction kit (QIAGEN) and then sequenced using the ABI PRISM 310 Genetic Analyser (PE Applied Biosystem). The ITS1/ITS4 amplification products were blotted onto nylon membranes and probed with T. magnatum specific oligonucleotides: TmagI, TmagII, ITSMAGN and ITSBACK3 (Amicucci et al. 1998; Rubini et al. 2001), according to the method described in Amicucci et al. (2002).
Morphological and molecular analyses The roots from each sample were washed in sterile distilled water and about 6500 root tips were examined under a dissecting stereomicroscope; the mycorrhizae with morphological features similar to those of Tuber species (Rauscher et al. 1995; Zambonelli et al. 1995, 1999) were separated and analysed using molecular methods (335 root tips). The anatomical structure of the mantles was examined under a Zeiss 40 light microscope and images were captured using a highresolution, colour video camera (JVC, Yokohama, Japan). The total genomic DNA was extracted from single or pooled mycorrhizal root tips as described by Paolocci et al. (1999). DNA from fruitbodies of known Tuber species was extracted from about 200 mg of tissue using the Lee and Taylor (1990) method. The amplification experiments were performed in a PTC-200 DNA Engine (Genenco) Peltier Thermal Cycler. The Internal Transcribed Spacers (ITS)
Results and discussion In this study we used molecular techniques to study the T. magnatum ectomycorrhizal community in natural T. magnatum ‘‘truffie `re’’ and were able to identify T. magnatum mycorrhizae and other coexisting Tuber species. This is the first time molecular techniques have been used to identify these species in the field, hence this work constitutes an important step towards gaining a better understanding of the autoecology of T. magnatum, the most valuable truffle species. Until recently, such studies in the field have relied on morphological identification of the mycorrhizae (Gregori et al. 1990). However, this method of identification is highly complex and often impossible to apply because in a natural environment mycorrhizae are found at different stages of development and lack of some diagnostic morphological features (Selosse 2001). In the investigated root samples we found approximately
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L. Bertini et al.
6500 mycorrhizal tips located almost exclusively in the top 15 cm of the soil, which is probably where mineralization processes are most active, and only rarely were mycorrhizae found at greater depths. A total of 335 ectomycorrhizae with Tuber-like morphological features (white to ochre colour, with or without cystidia and pseudoparenchymatous mantles with epidermoid cells), Fig. 1, were selected from all the mycorrhizae collected for molecular analysis. A morphological approach would only have permitted the grouping of these mycorrhizae into Tuber-like mycorrhizal morphotypes, which do not always represent a single species. Only through the use of molecular tools was it possible to identify them unequivocally. All the samples were analysed using the ITS–RFLP method and the T. dryophilum, T. maculatum and T. magnatum species were identified (Fig. 2). Hybridization experiments confirmed the data obtained by RFLP analysis, allowing the unequivocal identification of the T. magnatum ectomycorrhizal samples, as shown in Fig. 3. Other species,
for which no RFLP diagnostic profiles were available, were analysed by sequencing the ITS region. Some samples containing more than one mycorrhizal tip, collected in a single mycorrhizal root, and morphologically classified as Tuber-like, unexpectedly yielded two different amplification fragments when amplified with the primers ITS5/ ITS6 and ITS1/ITS4. The sequence of both the amplification products revealed the coexistence of two different ectomycorrhizal species (Fig. 4). This finding confirms the inherent limitations of the morphological method in the identification of Tuber ectomycorrhizae while demonstrating the value of molecular methods in identifying mycorrhizae collected in the field. Molecular methods allow us to analyse single mycorrhized root tips and, in some cases, to make a selective amplification in mixtures containing different fungal species or in mixtures of plant and fungal DNA. The molecular screening of the mycorrhizae morphologically classified as Tuber-like revealed that a high percentage actually belonged to Tuber
Figure 1. Pseudoparenchymatous mantles composed of epidermoid cells from Tuber maculatum ectomycorrhiza.
Figure 3. Hybridization with T. magnatum specific oligonucleotide (TmagI). Lanes 1–12, Tuber-like morphotypes; lane 13, positive control: T. magnatum fruitbody.
Figure 2. RFLP analysis of ITS region (primers ITS1–4) using restriction enzymes Rsa I (A) and Taq I (B). M: molecular size marker (GeneRulerTM 100 bp DNA Ladder Plus); lanes 1–8, ectomycorrhizal T. magnatum-like morphotypes; lane 9, T. magnatum fruiting body.
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Acknowledgements This study was supported by the ‘‘Regione Marche’’ CEE 2081/93 DOCUP Ob. 5b-Misura 1.1.3.Azione 2: Sperimentazione e tartuficoltura. We wish to thank the truffle-seller for allowing us to study the ‘‘truffie `re’’ known as ‘‘Il Colle’’. We also wish to thank Dr. Ian Hall (New Zealand Institute for Crop Food Research Limited, Invermay Agriculture Centre, New Zealand), for his helpful suggestions.
References Figure 4. ITS amplification products obtained with ITS5–ITS6 primers. M: molecular size marker (GeneRulerTM 100 bp DNA Ladder); lanes 1–7, Tuber-like morphotypes identified by sequence analyses: lane 1, Tuber maculatum; lane 2A, Tuber maculatum; lane 2B, Tuber rapedorum; lane 3, Tuber ferrugineum; lane 4, lane 4A Tuber ferrugineum, lane 4B no similarity found; lane 5, Tuber ferrugineum; lane 6 T. dryophilum.
species usually considered contaminating when found in the greenhouse or ‘‘truffie `re’’ (Tibiletti and Zambonelli 1999). Of the 335 tips examined T. dryophilum, T. rufum and, above all, T. maculatum ectomycorrhizae were present in the following percentages: 9.6%, 19.8% and 48.9%, respectively. Although these species are considered contaminants in the truffle cultivation of T. magnatum, the results obtained suggest that a biological equilibrium exists in nature (Pirazzi 2001). The percentage of T. magnatum ectomycorrhizae detected was low, about 4.4%. This result, however, is not surprising because certain species, which commonly form mycorrhizae, are poorly, or not at all, represented in the above-ground fruiting record (Jonson et al. 1999; Dahlberg 2001). The large biomass of Tuber fruiting bodies in the ‘‘truffie `re’’ despite limited ectomycorrhizal biomass in the soil may be explained by the saprotrophic ability of the ascoma. The ascoma becomes independent from the host plant at an early stage of its development and sustains its growth with nutrients obtained directly from the soil, as reported for T. uncinatum and T. melanosporum (Barry et al. 1993). Further studies, molecular as well as ecological, should be carried out in other ‘‘truffie `re’’ sites to identify those selective physical and biological factors that regulate T. magnatum symbiosis in a natural environment. Such information could prove invaluable to the successful cultivation of this species.
Amicucci, A., Zambonelli, A., Giomaro, G., Potenza, L., Stocchi, V., 1998. Identification of ectomycorrhizal fungi of the genus Tuber by species-specific ITS primers. Mol. Ecol. 7, 273–277. Amicucci, A., Zambonelli, A., Guidi, C., Stocchi, V., 2001. Morphological and molecular characterization of Pulvinula constellatio ectomycorrhizae. FEMS Microbiol. Lett. 194, 121–125. Amicucci, A., Guidi, C., Potenza, L., Stocchi, V., 2002. Microsatellite primed-PCR to select molecular markers for Tuber species. Biotechnol. Lett. 24, 263–267. Barry, D., Staunton, S., Callot, G., 1993. Mode of the absorption of water and nutrients by ascocarps of Tuber melanosporum and Tuber aestivum: a radioactive tracer technique. Can. J. Bot. 72, 317–322. Bertini, L., Potenza, L., Zambonelli, A., Amicucci, A., Stocchi, V., 1998. Restriction fragment length polymorphism species–specific patterns in the identification of white truffles. FEMS Microbiol. Lett. 164, 397–401. Bertini, L., Amicucci, A., Agostini, D., Polidori, E., Potenza, L., Guidi, C., Stocchi, V., 1999. A new pair of primers designed for amplification of the ITS region in Tuber species. FEMS Microbiol. Lett. 173, 239–245. Bocci, R., 1982. Lineamenti climatici delle Marche. Quaderni per l’Ambiente della Regione Marche. Danielson, R.N., 1984. Ectomycorrhiza formation by the operculate discomycete Sphaerosporella brunnea (Pezizales). Mycologia 76 (3), 454–461. Dahlberg, A., 2001. Community ecology of ectomycorrhizal fungi: an advancing interdisciplinary field. New Phytol. 150, 555–562. Donnini, D., Bencivenga, M., Baciarelli-Falini, L., 2000. Risultati di esperienze pluriennali nella coltivazione di Tuber magnatum Pico in Umbria. Micol. Ital. 29 (3), 33–39. Hall, I.R., Zambonelli, A., Primavera, F., 1998. Ectomycorrhizal fungi with edible fruiting bodies, Tuber magnatum Tuberaceae. Econ. Bot. 52, 192–200. Gregori, G.L., LoBue, G., Maggiorotto, G., 1990. Caratterizzazione di ectomicorrize in tartufaie naturali e di impianto e valutazione della loro competitivita ` nei confronti di Tuber magnatum. In: Bencivenga, M., Granetti, B. (Eds.), Atti del II Congresso Internazionale
ARTICLE IN PRESS 64 sul Tartufo, Spoleto, 24–27 Novembre 1988, pp. 219–246. Gregori, G., Cecchini, M., Elisei, S., Pasquini, L., Sacchi, A., Spezi, D., 2001. Tartufaie controllate di tuber magnatum: prove di miglioramento. In: Fe´de´ration., Franc˛aise des Trufficulteurs (Ed.), Actes du V1 Congre `s International Science et culture de la truffe. Aix en provence 4–9 March 1999, pp. 394–399. Gregori, G., 2002. Problems and expectations with the cultivation of Tuber magnatum. In: Hall, I., Wang, Y., Danell, E., Zambonelli, A. (Eds.), Proceedings of the Second International Workshop on Edible Ectomycorrhizal Mushrooms. 3–6 July 2001. Institute for Crop & Food Research Limited (CD room), Christchurch, N.Z.: New Zealand. Jonson, L., Dahlberg, A., Nilsson, M.C., Zackrisson, O., Kare ´n, O., 1999. Ectomycorrhizal fungal communities in late-successional Swedish boreal forests and composition following wildfire. Mol. Ecol. 8, 205–217. Lee, S.L., Taylor, J.W., 1990. Isolation of DNA from fungal mycelia and single spores. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocol, a Guide to Methods and Applications. Academic Press, New York, pp. 282–287. Paolocci, F., Rubini, A., Granetti, B., Arcioni, S., 1999. Rapid molecular approach for a reliable identification of Tuber spp. ectomycorrhizae. FEMS Microbiol. Ecol. 28, 23–30. Pirazzi, R., 2001. Tuber magnatum Pico: un fungo micorrizogeno tardivo. In: Fe´de´ration., Franc˛aise des Trufficulteurs (Ed.), Actes du V1 Congre `s International
L. Bertini et al. Science et culture de la truffe. Aix en Provence 4–9 March 1999, pp. 233–236. Rauscher, T, Agerer, R., Chevalier, G., 1995. Ektomycorrhizen von Tuber melanosporum, Tuber mesentericum und Tuber rufum (Tuberales) an Corylus avellana L. Nova Hedwigia 61, 282–322. Rubini, A., Paolocci, F., Granetti, B., Arcioni, S., 2001. Morphological characterization of molecular-typed Tuber magnatum ectomycorrhizae. Mycorrhiza 11, 179–185. Selosse, M.A., 2001. Apport des techniques ge ´ne´tiques et mole´culaires `a la connaissance des communaute´s et des comunitys de champignons ectomycorhiziens. Lejeunia 165, 2–107. Tibiletti, E., Zambonelli, A., 1999. I tartufi della provincia di Forlı´-Cesena. Pa ` tron, Bologna, Italy. White, T.J., Bruns, T, Lee, S.L., Taylor, J.W., 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR Protocol, a Guide to Methods and Applications. Academic Press, New York, pp. 315–322. Zambonelli, A., Salomoni, S., Pisi, A., 1995. Caratterizzazione anatomo-morfologica e micromorfologica delle micorrize di Tuber borchii Vitt., Tuber aestivum Vitt., Tuber mesentericum Vitt., Tuber brumale Vitt., Tuber melanosporum Vitt. su Pinus pinea L. Micol. Ital. 24, 119–137. Zambonelli, A., Iotti, M., Amicucci, A., Pisi, A., 1999. Caratterizzazione anatomo-morfologica delle micorrize di Tuber maculatum Vittad. su Ostrya carpinifolia Scop. Micol. Ital. 28, 29–35.
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Molecular identification of Tuber magnatum ectomycorrhizae in the field Luana Bertinia, Ismaela Rossia, Alessandra Zambonellib, Antonella Amicuccia, Achille Sacchic, Matteo Cecchinid, Gianluigi Gregoric, Vilberto Stocchia, a
Istituto di Chimica Biologica Giorgio Fornaini, Universita ` degli Studi di Urbino, Carlo Bo, Via Saffi 2, 61029 Urbino (PU), Italy b Dipartimento di Protezione e Valorizzazione Agroalimentare, Universita ` di Bologna, Via Fanin 46, 40127 Bologna, Italy c Centro Sperimentale di Tartuficoltura, Regione Marche, Via Macina 2, 61048 Sant’Angelo in Vado (PU), Italy d Istituto e Orto Botanico, Cattedra di Botanica, Universita ` degli Studi di Urbino, Via Bramante 28, 61029 Urbino (PU), Italy. Accepted 8 June 2005
KEYWORDS Tuber magnatum; Ectomycorrhizae; Molecular identification; ‘‘truffie `re’’
Summary Tuber ectomycorrhizae in a Tuber magnatum ‘‘truffie `re’’, located in Central Italy, were studied using molecular methods. Specifically, RFLP–ITS analyses, ITS sequencing and specific probes hybridization were used to identify 335 Tuber-like ectomycorrhizal morphotypes. Molecular identification was possible even when distinct morphological characteristics were lacking. For the first time, T. magnatum ectomycorrhizae and other coexisting Tuber species collected in the field were analysed using molecular tools for unambiguous identification. Although the ‘‘truffie `re’’ under investigation yields good harvests of T. magnatum fruiting bodies, the percentage of T. magnatum ectomycorrhizae found was very low (less than 4.4% of the 335 root tips analysed), whereas the percentages of Tuber maculatum and Tuber rufum were considerably higher (48.9% and 19.0%, respectively). & 2005 Elsevier GmbH. All rights reserved.
Corresponding author. Tel.: +39 0722 305262; fax: +39 0722 320188.
E-mail address: [email protected] (V. Stocchi). 0944-5013/$ - see front matter & 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2005.06.003
ARTICLE IN PRESS 60
L. Bertini et al.
Introduction Truffles are the fruiting bodies of ascomycetous fungi belonging to the genus Tuber, which form ectomycorrhizae on the roots of angiosperms and gymnosperms. Some Tuber species, such as Tuber magnatum, produce edible fruiting bodies. T. magnatum is the most highly regarded truffle, with a market demand far exceeding the quantities that can be harvested from its natural wild habitats, primarily in Italy and Istria (Croatia) (Hall et al. 1998). Unlike Tuber melanosporum, Tuber aestivum and Tuber borchii, T. magnatum has not been successfully cultivated in truffle beds (Tibiletti and Zambonelli 1999). This is partly because it is difficult to obtain plants infected with T. magnatum that are uncontaminated by other species of white truffle, such as T. maculatum and T. borchii, or Sphaerosporella brunnea (Danielson 1984) and Pulvinula constellatio (Amicucci et al. 2001). T. maculatum, T. borchii and other Tuber species also seem to actively compete with T. magnatum when artificially infected plants are planted in ‘‘truffie `res’’ (Donnini et al. 2000). In order to improve T. magnatum cultivation and to identify the specific requirements of this fungus, many ecological studies have been carried out in natural ‘‘truffie `res’’ (Hall et al. 1998). However, very few studies have been conducted on the ectomycorrhizal communities of these productive areas. In all previous investigations mycorrhizae were identified using morphological methods,
Table 1.
which are imprecise and do not allow us to unequivocally distinguish T. magnatum mycorrhizae from those of T. maculatum, T. dryophilum and T. borchii (Gregori 2002). The present study uses molecular methods to investigate the ectomycorrhizal communities of different Tuber species in a productive T. magnatum ‘‘truffie `re’’.
Materials and methods Geographical location and ecological characteristics of the natural ‘‘truffie `re The ‘‘truffie `re’’ under investigation is located in Central Italy in the Marches region, in an area in the province of Pesaro-Urbino known as ‘‘Il Colle’’, situated 270 m a.s.l., at 431460 4300 latitude and 121450 2000 longitude. Covering about 6 ha, the ‘‘truffie `re’’ lies in a cool shady valley, exposed to the east. The climate is sub-Mediterranean and rainfall usually exceeds 900 mm per year mainly falling around the spring and autumn equinoxes with a high peak in the autumn and a second lower peak in the spring typical of Adriatic inland areas (Bocci 1982). The physical–chemical characteristics of the soil in the ‘‘truffie `re’’ are reported in Table 1. The ‘‘truffie `re’’ is almost completely covered by vegetation (about 95%), consisting of herbaceous (60%), shrub (18%) and arboreal species (22%). The T. magnatum host plants are oaks, poplars, willows
Physical–chemical characteristics of soil
Parameters
Average
Maximum values
Minimum values
Standard deviation
Sand (g/kg) Mud (g/kg) Clay (g/kg) pH (H2O) pH (KCl) Total carbonate (g/kg) Active calcium carbonate (g/kg) Organic matter (g/kg) Total nitrogen (g/kg) Phosphate (assimilable) (mg/kg) Potassium (exchangeable) (mg/kg) Magnesium (exchangeable) (mg/kg) Cation exchange capacity (meq/100 g) Iron (assimilable) (mg/kg) Manganese (assimilable) (mg/kg) Zinc (assimilable) (mg/kg) Copper (assimilable) (mg/kg)
354.0 470.7 175.3 8.0 7.6 201.5 45.4 27.4 1.6 4.0 271.6 392.7 27.4 22.8 19.4 2.7 2.5
505.0 589.0 298.0 8.5 8.1 280.0 101.0 46.7 2.5 8.0 520.0 504.0 34.8 41.5 26.6 4.6 3.5
134.0 380.0 105.0 7.6 7.3 160.0 24.0 15.7 1.1 1.0 160.0 321.0 21.1 10.4 11.1 1.9 1.5
116.0 77.7 63.8 0.2 0.2 2.8 24.8 11.6 0.5 2.2 106.1 69.0 4.7 9.1 6.1 0.9 0.7
ARTICLE IN PRESS Molecular identification of Tuber magnatum ectomycorrhizae in the field Table 2.
61
Representative plant species present in ‘‘truffie `re’’ under study
Arboreal species
Shrub species
Herbaceous vegetation
Ostrya carpinifolia Populus alba Populus nigra Populus tremula Quercus pubescens Salix alba Salix caprea
Clematis vitalba Cornus sanguinea Euonymus europaeus Hedera helix Prunus spinosa Rubus caesius Rubus ulmifolius
Brachypodium sylvaticum Equisetum arvense Equisetum telmateja Eupatorium cannabinum Euphorbia cyparissias Pteridium aquilinum Ranunculus acris Symphytum tuberosum Tamus communis
and hornbeam (Table 2), and the ‘‘truffie `re’’ has produced about 15 kg of truffles per hectare over the last 5 years (Gregori et al. 2002).
Sample collection The soil samples were collected using a 60 mm diameter soil corer at depths of 0–15 cm, 15–30 cm and 30–45 cm at three different times in 2001: May (2 May, 12 May), June–July (28 June, 5 July, 12 July) and September–October (7 September, 15 September, 16 October). A total of 90 soil samples were taken. Sampling was done only in the more productive areas in order to increase the probability of finding natural T. magnatum ectomycorrhizae. The soil samples were stored overnight at 4 1C and were separated from the soil using a 0.5 mm mesh sieve.
regions were amplified with the primers ITS1/ ITS4, ITS1/ITS2, ITS5/ITS6 (White et al. 1990; Bertini et al. 1999). The primers were synthesized by Sigma-Aldrich (Poole, UK). The RFLP (Restriction Fragment Length Polymorphism) analyses with Taq I and Rsa I enzymes (Takara Biomedicals) of the amplified ITS 5–6 region were performed on unpurified PCR products (Bertini et al. 1998). The amplified ITS 5–6 region was first purified with the QIAquick PCR purification kit or with the gel extraction kit (QIAGEN) and then sequenced using the ABI PRISM 310 Genetic Analyser (PE Applied Biosystem). The ITS1/ITS4 amplification products were blotted onto nylon membranes and probed with T. magnatum specific oligonucleotides: TmagI, TmagII, ITSMAGN and ITSBACK3 (Amicucci et al. 1998; Rubini et al. 2001), according to the method described in Amicucci et al. (2002).
Morphological and molecular analyses The roots from each sample were washed in sterile distilled water and about 6500 root tips were examined under a dissecting stereomicroscope; the mycorrhizae with morphological features similar to those of Tuber species (Rauscher et al. 1995; Zambonelli et al. 1995, 1999) were separated and analysed using molecular methods (335 root tips). The anatomical structure of the mantles was examined under a Zeiss 40 light microscope and images were captured using a highresolution, colour video camera (JVC, Yokohama, Japan). The total genomic DNA was extracted from single or pooled mycorrhizal root tips as described by Paolocci et al. (1999). DNA from fruitbodies of known Tuber species was extracted from about 200 mg of tissue using the Lee and Taylor (1990) method. The amplification experiments were performed in a PTC-200 DNA Engine (Genenco) Peltier Thermal Cycler. The Internal Transcribed Spacers (ITS)
Results and discussion In this study we used molecular techniques to study the T. magnatum ectomycorrhizal community in natural T. magnatum ‘‘truffie `re’’ and were able to identify T. magnatum mycorrhizae and other coexisting Tuber species. This is the first time molecular techniques have been used to identify these species in the field, hence this work constitutes an important step towards gaining a better understanding of the autoecology of T. magnatum, the most valuable truffle species. Until recently, such studies in the field have relied on morphological identification of the mycorrhizae (Gregori et al. 1990). However, this method of identification is highly complex and often impossible to apply because in a natural environment mycorrhizae are found at different stages of development and lack of some diagnostic morphological features (Selosse 2001). In the investigated root samples we found approximately
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6500 mycorrhizal tips located almost exclusively in the top 15 cm of the soil, which is probably where mineralization processes are most active, and only rarely were mycorrhizae found at greater depths. A total of 335 ectomycorrhizae with Tuber-like morphological features (white to ochre colour, with or without cystidia and pseudoparenchymatous mantles with epidermoid cells), Fig. 1, were selected from all the mycorrhizae collected for molecular analysis. A morphological approach would only have permitted the grouping of these mycorrhizae into Tuber-like mycorrhizal morphotypes, which do not always represent a single species. Only through the use of molecular tools was it possible to identify them unequivocally. All the samples were analysed using the ITS–RFLP method and the T. dryophilum, T. maculatum and T. magnatum species were identified (Fig. 2). Hybridization experiments confirmed the data obtained by RFLP analysis, allowing the unequivocal identification of the T. magnatum ectomycorrhizal samples, as shown in Fig. 3. Other species,
for which no RFLP diagnostic profiles were available, were analysed by sequencing the ITS region. Some samples containing more than one mycorrhizal tip, collected in a single mycorrhizal root, and morphologically classified as Tuber-like, unexpectedly yielded two different amplification fragments when amplified with the primers ITS5/ ITS6 and ITS1/ITS4. The sequence of both the amplification products revealed the coexistence of two different ectomycorrhizal species (Fig. 4). This finding confirms the inherent limitations of the morphological method in the identification of Tuber ectomycorrhizae while demonstrating the value of molecular methods in identifying mycorrhizae collected in the field. Molecular methods allow us to analyse single mycorrhized root tips and, in some cases, to make a selective amplification in mixtures containing different fungal species or in mixtures of plant and fungal DNA. The molecular screening of the mycorrhizae morphologically classified as Tuber-like revealed that a high percentage actually belonged to Tuber
Figure 1. Pseudoparenchymatous mantles composed of epidermoid cells from Tuber maculatum ectomycorrhiza.
Figure 3. Hybridization with T. magnatum specific oligonucleotide (TmagI). Lanes 1–12, Tuber-like morphotypes; lane 13, positive control: T. magnatum fruitbody.
Figure 2. RFLP analysis of ITS region (primers ITS1–4) using restriction enzymes Rsa I (A) and Taq I (B). M: molecular size marker (GeneRulerTM 100 bp DNA Ladder Plus); lanes 1–8, ectomycorrhizal T. magnatum-like morphotypes; lane 9, T. magnatum fruiting body.
ARTICLE IN PRESS Molecular identification of Tuber magnatum ectomycorrhizae in the field
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Acknowledgements This study was supported by the ‘‘Regione Marche’’ CEE 2081/93 DOCUP Ob. 5b-Misura 1.1.3.Azione 2: Sperimentazione e tartuficoltura. We wish to thank the truffle-seller for allowing us to study the ‘‘truffie `re’’ known as ‘‘Il Colle’’. We also wish to thank Dr. Ian Hall (New Zealand Institute for Crop Food Research Limited, Invermay Agriculture Centre, New Zealand), for his helpful suggestions.
References Figure 4. ITS amplification products obtained with ITS5–ITS6 primers. M: molecular size marker (GeneRulerTM 100 bp DNA Ladder); lanes 1–7, Tuber-like morphotypes identified by sequence analyses: lane 1, Tuber maculatum; lane 2A, Tuber maculatum; lane 2B, Tuber rapedorum; lane 3, Tuber ferrugineum; lane 4, lane 4A Tuber ferrugineum, lane 4B no similarity found; lane 5, Tuber ferrugineum; lane 6 T. dryophilum.
species usually considered contaminating when found in the greenhouse or ‘‘truffie `re’’ (Tibiletti and Zambonelli 1999). Of the 335 tips examined T. dryophilum, T. rufum and, above all, T. maculatum ectomycorrhizae were present in the following percentages: 9.6%, 19.8% and 48.9%, respectively. Although these species are considered contaminants in the truffle cultivation of T. magnatum, the results obtained suggest that a biological equilibrium exists in nature (Pirazzi 2001). The percentage of T. magnatum ectomycorrhizae detected was low, about 4.4%. This result, however, is not surprising because certain species, which commonly form mycorrhizae, are poorly, or not at all, represented in the above-ground fruiting record (Jonson et al. 1999; Dahlberg 2001). The large biomass of Tuber fruiting bodies in the ‘‘truffie `re’’ despite limited ectomycorrhizal biomass in the soil may be explained by the saprotrophic ability of the ascoma. The ascoma becomes independent from the host plant at an early stage of its development and sustains its growth with nutrients obtained directly from the soil, as reported for T. uncinatum and T. melanosporum (Barry et al. 1993). Further studies, molecular as well as ecological, should be carried out in other ‘‘truffie `re’’ sites to identify those selective physical and biological factors that regulate T. magnatum symbiosis in a natural environment. Such information could prove invaluable to the successful cultivation of this species.
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