Morphological and molecular typing of the below-ground fungal community in a natural Tuber magnatum truffle-ground

Authors

  • Claude Murat,

    1. UMR INRA/UHP 1136 “Interactions Arbres/Micro-Organismes”, Centre INRA de Nancy, F-54280 Champenoux, France
    2. Dipartimento di Biologia Vegetale dell'Università di Torino, Viale Mattioli, 25, 10125-Torino, Italy
    Search for more papers by this author
    • 1

      These authors contributed equally to this work.

  • Alfredo Vizzini,

    1. Dipartimento di Biologia Vegetale dell'Università di Torino, Viale Mattioli, 25, 10125-Torino, Italy
    Search for more papers by this author
    • 1

      These authors contributed equally to this work.

  • Paola Bonfante,

    1. Dipartimento di Biologia Vegetale dell'Università di Torino, Viale Mattioli, 25, 10125-Torino, Italy
    2. Istituto per la Protezione delle Piante del CNR, Sezione di Torino, Italy
    Search for more papers by this author
  • Antonietta Mello

    Corresponding author
    1. Istituto per la Protezione delle Piante del CNR, Sezione di Torino, Italy
    Search for more papers by this author

  • Edited by G.M. Gadd

*Corresponding author. Tel.: +39 0116502927; fax: +39 0116705962, E-mail address: a.mello@ipp.cnr.it

Abstract

The aims of the work were to elucidate the distribution of the ectomycorrhizal fungus Tuber magnatum Pico during its symbiotic stage, and to identify the root-associated fungi in a natural truffle-ground located in North Italy. Ectomycorrhizal root tips were harvested in the truffle ground, sorted in morphotypes and analyzed by ITS. Morphological and molecular analyses revealed that (i) T. magnatum mycorrhizae were rare and independent on the fruitbody productions and (ii) the dominant fungal species belonged to Thelephoraceae, followed by Tuberaceae and Sebacinaceae.

1Introduction

Truffles are ascomycetous fungi that form ectomycorrhizae (ECM) with the roots of trees, such as oak, poplar, willow, hazel [1], and shrubs, such as Cistus[2]. Some of them are products in great demand on the food market in many countries. Tuber magnatum Pico, commonly known as the white truffle of Alba (http://www.truffle.org), is of special interest. Its unique taste and fragrance, as well as its limited availability make T. magnatum fruitbodies one of the most expensive delicacies on the market. While T. melanosporum Vittad. is generally sold at € 30–40/100 g in France, T. magnatum reached € 300–400/100 g in Fall 2003. A productive truffle-ground represents therefore a conspicuous economic source for rural communities. Knowledge on the microbial environment where truffles develop is surely a pre-requisite for a better exploitation of the natural truffle-grounds. However, data on truffle ectomycorrhizae ecology are very limited, previous studies being mainly focussed on truffle mycorrhizal identification, either with isoenzymes [3] or molecular tools [4–9]. Advances on Tuber spp. cultivation have allowed the development of mycorrhizal seedlings with some truffle species. T. melanosporum was the first truffle produced in experimental fields using inoculated seedlings [10]. These cultural techniques are available for T. uncinatum[11] and T. borchii[12]; however controlled production of T. magnatum is not possible yet.

T. magnatum ectomycorrhizae were identified only one time in wild samples by using multilocus electrophoresis [3]. Specific primers, based on the T. magnatum internal transcribed spacer (ITS) of the ribosomal DNA sequence, were used for a molecular characterization of mycorrhizal seedlings raised under controlled conditions [8,9]. These molecular probes also enabled a new morphological characterization to be formulated for T. magnatum mycorrhizae.

Many questions, which are at the basis of our knowledge on the genetic diversity and distribution of ectomycorrhizal fungi in natural ecosystems, have been recently listed [13]. Notwithstanding an abundant literature, all these questions are still fully open as far as concerns truffle ecology: (i) how abundant are truffle mycorrhizae in a natural truffle ground? (ii) does their presence match the fruitbody production? (iii) as a consequence, can we detect mycorrhizae exclusively in productive zones, or also in non-productive ones? (iv) which other ectomycorrhizal fungi are present in a truffle-ground?

The simplest method to investigate a fungal species during its symbiotic phase, and within the ectomycorrhizal community, is the morphological typing of mycorrhizae in nature. This method, however, is dependent on many factors, as the age, the host tree species and environmental conditions. For this reason, morphological typing of ectomycorrhizae is currently supported by molecular methods.

The ITS of the ribosomal DNA has been mostly used to identify species in subterranean ECM fungal community. ITS sequencing followed from a search of the unknown sequences in ITS nucleotide databases has been broadly used [16–18]. Here, we aimed to elucidate the white truffle distribution during its symbiotic stage and to identify fungi associated with T. magnatum in a natural truffle-ground. We took advantage on our previous work, where we mapped two genotypes of T. magnatum fruitbodies collected in the natural truffle-ground of Montemagno (Piedmont, Italy) revealing productive and not productive areas [19]. As a second step, mycorrhiza samples were harvested, sorted into morphotypes and analyzed by ITS. Even if the sampling was not extensive and many cautions had to be taken in digging the soil of the productive area under owner scrutiny, this work represents the first study on the Tuber symbiotic phase in a truffle-ground. The comparison between the morphological and the molecular analyses allowed us to demonstrate that (i) the T. magnatum mycorrhizae are very rare in a productive truffle-ground, (ii) there were no clear linkages between the temporal/spatial productivity of a single plant and the presence of T. magnatum mycorrhizae, and (iii) the dominant fungal family in the subterranean ECM community is represented by the Thelephoraceae.

2Materials and methods

2.1Identification of the truffle-ground, geographical data

The truffle-ground, of about 7.000 m2, is placed in a valley in Montemagno (Asti, Piedmont, North Italy, 8°19′35″4 East, 44°59′2″40 North), as described in a previous work, where the production of the white truffle was followed during the winters 1997–2002 [19]. Twenty-one root samples were collected: 12 samples were collected in November (10 in productive and 2 in not productive areas), when it is period of the harvest of the fruibodies and 9 in May (6 in productive and 3 in not productive areas), when the harvesting is over (Table 1). The sampling position was mapped in respect to a chosen tree (Fig. 1, Table 1). When possible, up to three samples were collected within a small area (Table 1, Fig. 1). The limited number of samplings was due to the risk of breaking down the fungal web digging the soil of the productive truffle-ground.

Table 1.  List of (1) ectomycorrhizal morphotypes found in the samples (S), under productive (Prod.) and not productive (Not prod.); (2) fungi identified by ITS sequencing followed by blast searching
T. N.AreaFirst sampling in November 2001Second sampling in May 2002
SMorphotypesITS analysisSMorphotypesITS analysis  
  1. TN, indicates the tree number, –, indicates absence of data, * indicates sequences not deposited in NCBI.

2Prod.1Unidentified1UnidentifiedTomentella/Thelephora (AJ879677)
      Tuber magnatumTuber magnatum (AJ879679), Helvella sp. (AJ879678)
   UnidentifiedEpicoccum sp. (AJ879641), Pezizaceae (AJ879640) Tuber sp.Tuber maculatum (AJ879680)
 Prod.2Tomentella sp.Tomentella sp. (AJ879642)4Tomentella sp.Tomentella sp. (AJ879688), Verticillium sp. (AJ879689), Dothideomycetes (AJ879690)
   Tuber maculatumTuber maculatum (AJ879643)   
 Prod.3Tomentella sp.Tomentella sp. (AJ879644)   
   UnidentifiedHelvella lacunosa (AJ879645), Tetracladium sp. (AJ879646), Tuber rufum (AJ879647)   
4Not prod.5Scleroderma sp.Scleroderma sp. (AJ879652)2Tuber sp.Helvella sp. (AJ879682)
   UnidentifiedStrumella sp. (AJ879653), Scleropezicula sp. (AJ879654), Sebacina sp. (AJ879655) Tomentella sp.Tomentella (AJ879681)
   UnidentifiedTomentella sp. (AJ879656)3Tomentella sp.Amphisphaeriaceae (AJ879684), Alternaria sp. (AJ879683)
   UnidentifiedSebacinaceae (AJ879657) UnidentifiedTuber magnatum (AJ879687), Sebacina incrustans (AJ879685), Helotiales (ericoids and endophytes) (AJ879686)
6Not prod.4UnidentifiedMortierella alpina (AJ879650), Fungal endophyte (AJ879648), Nectriaceae (AJ879649)   
   UnidentifiedTomentella sp. (AJ879651)   
   Tomentella sp.   
9Prod.6Tuber sp.Ericoid endophyte (Helotiales) (AJ879659), Nectriaceae (AJ879658)   
10Prod.10UnidentifiedSebacinaceae (AJ879661)   
11Prod.8Tomentella sp.Agaricales (AJ879660), Tomentella sp.*5No mycorrhizas 
 Prod.11Tuber sp.Agaricales (AJ879662)   
12Prod.12Tuber sp.Sebacinaceae (AJ879663)9UnidentifiedSebacinaceae (AJ879694)
   Tomentella sp.Tomentella sp. (AJ879664) Tomentella sp.
   Tuber sp.Nectria*, Tomentella sp. (AJ879665)7Unidentified
29Prod.13Tomentella sp.Tomentella sp. (AJ879666)Tomentella sp.Tomentella sp. (AJ879692) 
   UnidentifiedCortinariaceae (AJ879668),   
    Sebacinaceae (AJ879667)   
 Prod.14Tuber sp.Leptosphaeria sp. (AJ879672),8UnidentifiedTetracladium sp. (AJ879693)
    Nectria sp. (AJ879669), Sebacina epigea (AJ879670),   
    Tetracladium sp. (AJ879671), Pezizomycotina (AJ879673)   
   UnidentifiedCortinariaceae (AJ879675), Agaricales (AJ879674)   
   Tomentella sp.Tomentella sp. (AJ879488), Nectria sp. (AJ879676)   
31Not prod.   6Tuber sp.Tuber maculatum (AJ879691)
Figure 1.

Map of the truffle-ground located at Montemagno (Piedmont). Mycorrhizae samples collected in November 2001 are shown with blue numbers while those collected in May 2002 are shown with red numbers. The two zones delimited by circles indicate not productive areas.

2.2Morphological analysis

Twenty-one root samples were collected from 10-cm depth increments of the root system, rinsed in water and observed under a stereo dissecting microscope to look for ectomycorrhizal roots. A whole of 335 mycorrhizal root tips were sorted into 39 morphotypes on the basis of color, mantle shape and surface texture, presence of cystidia, and EM branching pattern [20,21]. When necessary, cross sections were made and examined under a light microscope for the presence of the Hartig net. Each morphotype grouped from 8 to 10 similar mycorrhizal tips. In two cases only 1 mycorrhizal tip was regarded as morphotype. Thirty-nine morphotypes (containing 335 mycorrhizal tips) were examined by morphological and molecular analyses.

2.3Molecular analysis

Total DNA was extracted from the identified 39 morphotypes (each containing from 8 to 10 similar mycorrhizal tips) by using the Dneasy Plant Mini Kit (Qiagen SA, Courtaboeuf, France) following the manufacturer's instructions. PCR reactions were performed with primers ITS1f and ITS4 [14,22]. rDNA libraries were produced by isolating the amplicons from gel, ligating them into the pGEM-T vector (Promega, Madison Wis.) and transforming the plasmids into competent JM109 cells. Bacterial colonies containing plasmids with rDNA inserts were identified by PCR. To sort the clones into groups the rDNA inserts were amplified by PCR, digested individually with two DNA restriction endonuclease treatments (HinfI, Sau3AI) and resolved on 2% agarose gels. Clones which showed different restriction patterns were sequenced. Similarities of the rDNA clones to sequences in the GenBank database were determined by using BLAST (NCBI). The result of each BLAST searching for the present paper was carefully examined. Among the listed organisms produced, those showing a low expected value (e) and a high score were assumed to be, with high probability, the species/genera which are present in the truffle-ground. Whenever we were not confident of the sequence attribution to a genus, the sequence was positioned in a higher taxon that could be the family or the order. As a consequence, only in few cases we were able to assign species by molecular analysis. Accession numbers are shown in Table 1.

3Results and discussion

This survey is the first attempt to study the ecology of the symbiotic phase of T. magnatum, by using molecular methods. Notwithstanding the drawback of having a limited number of samples (335 mycorrhizal tips from 21 samplings sorted in 39 morphotypes) our investigation allowed us to identify T. magnatum mycorrhizae in a natural truffle-ground. In addition, morphotyping and ITS sequencing of these mycorrhizal samples provided novel information on the ectomycorrhizal and endophytic species living in a T. magnatum truffle-ground (Table 1).

3.1A first glance at the subterranean fungal community

Morphological analysis allowed to distinguish 39 morphotypes; among them 23 were identified and 16 remained as Unidentified (Table 1). Morphotypes identities were confirmed in 14 out of 23 by the molecular analysis, while in 6 cases there was a discrepance between morphological and molecular data (Table 1). For these morphotypes, 4 out of 6 classified as Tuber, and 2 as Tomentella, the molecular analysis showed other taxa and, often, multiple fungal species. The explanation for the lack of correspondence in these cases can be due to: (i) the possibility that we missed some clones; (ii) the amount of DNA coming from endophytes, or common fungal contaminants, which are present around the selected morphotype, is greater and/or better amplificable than the DNA coming from the morphotype; (iii) similarity among morphotypes; (iv) a wrong morphological identification. In the remaining 3 cases the molecular analysis failed for technical problems.

The most frequent family, recognized by both morphological and molecular analyses, is constituted by Thelephoraceae (13 out of 39 samples, 32%) represented in all cases by Tomentella, except in one sample where Tomentella could not be distinguished from the close genus Thelephora (S1 in May 2002). The alignment of Tomentella sequences – which were found in our samplings – showed a broad heterogeneity, suggesting the presence of different species and revealing therefore the diversity occurring in the truffle-ground. A preliminary analysis of a T. melanosporum truffle-ground and a number of recent studies have indicated that tomentelloid fungi may be a widespread and important component of EM communities [30,23].

A second important group was represented by the order Pezizales (11 out of 39 morphotype). These comprises Pezizaceae, Helvellaceae (Helvella lacunosa), Sarcosomataceae (Strumella sp.) and Tuberaceae (T. magnatum, T. maculatum, T. rufum) (Table 1). They were mainly found in one part of the truffle-ground near trees 2 and 4 during the two seasons. For example, under the tree N 2: (1) T. magnatum was identified in May 2002, (2) T. rufum was found in November 2001, (3) T. maculatum was present in November, which is a fructification period, but also in May, (4) both H. lacunosa, present in November, and Helvella sp. found in May, were always associated to Tuber species. Interestingly enough, T. maculatum ascocarps have been harvested in this truffle-ground near trees 2 and 4, while no harvest of T. rufum ascocarps has been recorded so far (Gavazza, personal communication).

A third frequent family was represented by Sebacinaceae (in 8 cases associated to ectomycorrhizal tips).

Looking at the samplings under the tree N 4, Sebacina incrustans was detected on the unidentified morphotype where T. magnatum and ericoid and/or endophytic fungi belonging to Helotiales are present. Sebacina incrustans was identified as the fungal partner of one ectomycorrhizal sample [24], although Sebacinaceae are a family not so far considered in the major compilations of ectomycorrhizal taxa [1,20,21,25].

Our finding of a Sebacina sp. associated to T. magnatum ectomycorrhizae is in agreement with the discovery of sebacinoid ectomycorrhizae associated to ascomycetes ectomycorrhizae [24,26]. Nevertheless a close association between the ectomycorrhizae of Morchella (Pezizales) and those of one unidentified heterobasidiomycete was already reported [27,28] We assume that this heterobasidiomycete could belong to Sebacinaceae, because of the similarity of its dolipore ultrastructure (not perforated parenthesome) with that from this family.

This intimate association between species of mycorrhizal fungi is not new, in fact it is known that a single root tip may be colonized by multiple fungal species forming composite mycorrhizae as Suillus bovinusGomphidius roseus and Boletus edulisAmanita excelsa associations [21,29].

We found Sebacina epigea associated to a morphotype classified as Tuber, and other fungi belonging to the Sebacinaceae, always associated to unidentified morphotypes, except in one case when they are associated to a morphotype classified as Tuber. The absence of knowledge of the occurrence and the morphology of Sebacinoid ectomycorrhizae in 2001, at the beginning of our investigation could be a reason why Sebacinaceae are frequently found in unidentified morphotypes.

3.2Identification of T. magnatum mycorrhizae

In the screening of mycorrhizal tips, T. magnatum mycorrhizae were identified only twice. In one case, they were identified by morphological analysis and the identity confirmed by the ITS analysis (S1 May 2002; Table 1). In the second case the morphotype (consisted of old mycorrhizae) was unidentified and the identification was limited to the molecular analysis (S3 May 2002; Table 1). These results lead to the claim that T. magnatum mycorrhizae are very rare in a productive truffle-ground [19]: only two out of 39 samples, representing 5%. The low occurrence of a fungal species at the mycorrhizal level has been already reported [15]. It was found that Suillus pungens ECM root tips were rare in a Pinus muricata forest, whereas S. pungens sporocarps were abundant. In the same forest, Russula amoenolens root tips were abundant, whereas R. amoenolens sporocarps were rare. Macrofungi species which accounted for 70% of the annual fruiting biomass correspond to less than 30% of the colonized root tips [31]. Cortinarius was the most abundant genus forming 42.3% of sporocarps in a Pinus sylvestris stand at Riddarhhyttan, however belowground only 1.6% of the mycorrhizal tips examined could be attributed to this genus [32].

In both cases the two T. magnatum mycorrhizal samples were found during spring 2002, that it is a non-productive period for T. magnatum, and, in the second case, in a non-productive area (Fig. 1 and Table 1, near tree 4), suggesting that there is not a direct linkage between mycorrhizae and fruitbodies.

How could we explain the low frequence of T. magnatum mycorrhizae in a productive natural truffle-ground? The truffle life cycle is usually illustrated with three defined phases: a reproductive, a vegetative and a symbiotic phase [33]. However, the situation is surely more complex. A number of molecular and biochemical studies have shown the saprotrophic behavior of both the mycelium growing in pure culture and the fruitbodies of the T. borchii species [34–36]. A parasitic behavior of T. melanosporum towards grass was claimed [37]. Finally, T. excavatum and T. aestivum have been identified as endophytic fungi of achlorophillic orchids [38,39]. All these observations challenged the importance of the truffle symbiotic phase, suggesting by contrast that truffles may be more plastic in their metabolism than expected. They seem to move along differential nutritional strategies (saprotrophic, endophytic and symbiotic) depending on the environment and on the developmental phase of their life cycle.

The presence of different nutritional strategies has already been found in other fungi: the phytopathogenic Armillaria mellea forms mycorrhiza with Gastrodia[40] and Tricholoma matsutake could be at the very beginning symbiotic, than parasitic and finally saprotrophic [29].

In the case of T. magnatum, it seems that this fungus invests more in fruitbody formation than in root tips colonization. The condition of low mycorrhizal percentage and the spread and persistence of a large S. pungens genet fruiting abundantly were explained with three hypotheses [41]: (1) S. pungens is so efficient at obtaining C from its host that few mycorrhizal connections are needed for significant C transfer; (2) large genets visit the roots of more trees, pooling, consequently, more total C than smaller genets; (3) a fraction of the C is obtained saprotrophycally. The hypothesis that the few T. magnatum mycorrhizae are able to adsorbe nutrients from the plant in an extremely efficient way, does not seem us attractive, since T. magnatum mycorrhizae do not differentiate any mycelial cord. As an alternative, the association between plant and T. magnatum could not require a well differentiate mycorrhiza to establish a nutrient flow. Notwistanding the many papers illustrating the metabolisms which are active during the symbiosis [42] convincing evidences of a nutritional transfer among the partners are not available. Further analyses are surely required to improve knowledge on truffle ecosystems and, as a consequence, to enhance the success of experimental truffle production.

Acknowledgments

We wish to thank, Franco Ferraris, the owner of the truffle-ground at Montemagno, Virgilio Gavazza who kindly helped us during the samplings and Francis Martin for reading the manuscript. The research was funded by the National Council of Research in Italy, CRT Foundation (Cuneo, Italy) and CEBIOVEM (DM 17/10/2003, No. 193/2003). This project has benefited from a PhD fellowship from the Italian ISASUT (University of Torino) to Claude Murat.

Ancillary