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 , 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 bovinus–Gomphidius roseus and Boletus edulis–Amanita 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 : only two out of 39 samples, representing 5%. The low occurrence of a fungal species at the mycorrhizal level has been already reported . 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 . 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 .
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 . 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 . 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 and Tricholoma matsutake could be at the very beginning symbiotic, than parasitic and finally saprotrophic .
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 : (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  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.