Introduction

Many Tuber melanosporum Vittad. (Périgord black truffle) crops have a high carpophore production, but uncontrolled factors still induce a great deal of variability in production from year to year. Chevalier and Frochot (1997) and Lefevre and Hall (2001) indicate that contamination of truffières (truffle production sites) by other competing ectomycorrhizal fungi poses formidable problems for researchers working to optimize production of truffle species. Several authors have expressed their concern over the risk that commercial Chinese truffles (Tuber species) might be introduced accidentally or fraudulently into the plantations of Mediterranean truffles. Murat et al. (2008) have confirmed the appearance of T. indicum DNA in root tips and soil samples from a T. melanosporum plantation in Piedmont (Italy).

The international truffle market has seen regular shipments of Chinese truffles between China and Europe, Japan, United States, and Australia, for the last 14 years (Rey 2001; Wang and Hall 2001; Yamanaka et al. 2001; Yang 2001; García-Montero et al. 2005). Chinese truffles act as complements to the market for T. melanosporum because they are cheaper, have a milder aroma and the carpophores are of good quality for culinary purposes (Rey 2001).

New Chinese truffle species have been discovered in European markets, such as Tuber pseudoexcavatum Wang, G. Moreno, L. G. Riousset, J. L. Manjón, and G. Riousset (Wang et al. 1998). From the taxonomical point of view, T. pseudoexcavatum species were confirmed by several studies on phylogeny and taxonomy based on morphological characteristics, and DNA methods (Riousset et al. 2001; Zhang et al. 2005; Wang et al. 2006b). In the University of Alcalá, we synthesized T. pseudoexcavatum ectomycorrhizae using samples of fresh carpophores from the type collections. We observed that the ectomycorrhizae of this species had a similar morphology to the ectomycorrhizae of T. melanosporum (Manjón et al. 1998). However, we have not yet published a description of T. pseudoexcavatum mycorrhizae, as we are awaiting further information which would enable us to determine the taxonomy of the vast quantity of Chinese truffles that are being discovered and marketed.

Di Massimo et al. (1996), Zambonelli et al. (1996, 1997) and Comandini and Pacioni (1997) synthesized Tuber indicum Cooke and Massee ectomycorrhizae. They showed that these ectomycorrhizae also have a considerable affinity with T. melanosporum ectomycorrhizae. These authors, as well as García-Montero et al. (1997a, b, 2005) and Ferrara and Palenzona (2001), indicated the need to increase quality controls of mycorrhized plants in truffle culture, and warned that the Chinese truffles could displace truffles with a greater economic value and penetrate the Mediterranean Tuber ecosystems. In this regard, the latest trends for distinguishing the various Tuber ectomycorrhizae are based simultaneously on morphological taxonomical characters and a range of biochemical and DNA-based methods (Ferrara and Palenzona 2001; Mabru et al. 2001; Douet et al. 2004; Iotti and Zambonelli 2006).

It is, therefore, necessary to increase the current knowledge of the Chinese truffles ectomycorrhizae and to determine their ecological requirements and restrictions. We report the morphological characterization of T. pseudoexcavatum ectomycorrhizae, and we provide useful information for the rapid morphological screening of T. pseudoexcavatum and T. indicum ectomycorrhizae versus T. melanosporum ectomycorrhizae. We also determine whether T. pseudoexcavatum and T. indicum could be easily introduced into Mediterranean habitats of T. melanosporum, depending on the characteristics of the soils and host plants common to T. melanosporum. We did this by analyzing samples of soils inhabited by T. melanosporum in natural Spanish woods, and we used these soil samples to synthesize T. pseudoexcavatum and T. indicum ectomycorrhizae with the principal host plant of T. melanosporum in many Mediterranean areas, Quercus ilex L.

Materials and methods

Synthesis and study of Chinese truffles ectomycorrhizae

We synthesized T. indicum ectomycorrhizae using samples of fresh carpophores, and T. pseudoexcavatum ectomycorrhizae using samples of fresh carpophores from the same commercial batches as the type collections (isotypus AH-18383 and AH-18384). The taxonomical identifications are presented in Manjón et al. (1995) and Wang et al. (1998). The mycorrhizal synthesis of these Chinese species was done by applying a method of intensive mycorrhization on six Quercus ilex subsp. ballota (Desf.) Samp. plants, respectively, as described by Bencivenga (1982), but modified according to Manjón and García-Montero (1996). We also mycorrhized T. melanosporum with six plants as a comparative control test.

The Quercus ilex acorns and truffle carpophores were carefully washed, sterilized on the surface, and conserved in a fridge at a temperature of +4°C. The acorns were sown in semi-rigid plastic containers with a volume of 1,000 cc. The substrate used for seeding was a mixture of two calcareous soils from a natural T. melanosporum truffière, mixed with vermiculite and perlite, which was sterilized in an autoclave at 120°C for 4 h. At the moment of sowing, they were inoculated with a water suspension of ascospores obtained from finely-chopped carpophores. An acorn and a suspension containing 2 g of fruit body were placed in each container. The plants were kept under controlled environmental conditions in the Juan Carlos I Royal Botanical Garden at the University of Alcalá (Madrid, Spain). They were housed in a greenhouse with an average daily temperature of 20 to 25°C; relative humidity of 60 to 70%; watered by micro-sprinkler between one and three times a day for 1–3 min depending on the time of year; and under natural light conditions, with no artificial lighting. These parameters were controlled automatically using a computer. The plants were not fertilized. We obtained a batch of 18 well-lined, disease-free plants, with an average height of 17 cm and a diameter at the base of the plant of 2 cm.

The degree of mycorrhization of each plant was expressed as the number of Tuber mycorrhizae of the total number of apices counted, according to Bencivenga et al. (1987). The ectomycorrhizae were identified with a stereoscopic microscope (photo-Leica WildMZ8) and a microscope (photo-Leica LeitzDMRB) following the terms, descriptions, and recommendations of Agerer (1987–2002), Bencivenga et al. (1995) and Granetti (1995). This study was done with the criterion of selecting the macroscopic and microscopic characteristics of these ectomycorrhizae which would be most useful in the control procedures for mycorrhized plants in truffle culture, according to Bencivenga et al. (1987) and Granetti (1995). We, therefore, observed the morphological characters of the ectomycorrhizae, such as shape, size, type of ramifications, color, and mantle surface. The ectomycorrhizal color was described following Munsell (1976) standard soil color charts. For the analysis of the anatomical characters, we chose the median parts of adult ectomycorrhizae, as indicated by Agerer (1987–2002) and we observed the outer mantle surface and the mycelium. We measured the different characters, and maximum and minimum ranges of values were established by taking the necessary measurements until regularity was attained in the values observed, with a minimum of 30 measurements.

The morphological characteristics of T. indicum, and T. melanosporum ectomycorrhizae were described within the ranges and descriptions proposed by Granetti (1995), Comandini and Pacioni (1997), Zambonelli et al. (1997) and Granetti et al. (2005).

Soil analysis

In the mycorrhizal synthesis of the Chinese truffles, we used soil samples from natural Spanish woods inhabited by T. melanosporum. We took the soil samples from around a T. melanosporum truffière associated to Quercus ilex subsp. ballota and Q. faginea Lam., with a maximum carpophore production of up to 5,000 g/year. A more detailed description of the study area, the soils and the truffle habitats can be found in García-Montero et al. (2006).

We analyzed two soil samples. One soil sample was taken from the bare soil in the interior of this truffière, and the other at a distance of 5 m from the truffière. Only the first 30 cm of soil profile were taken and studied, as T. melanosporum usually bears fruit in this range. Sampling was done according to the FAO (1990). The following soil determinations were made: pH, total organic carbon (T.O.C.), total calcium carbonate (equivalent calcium carbonate), granulometric analysis, cation exchange capacity (C.E.C.) and the exchangeable cation saturation percentage (% V), following the methods of the ISRIC (1995); the textures are classified according to the International Society of Soil Science (I.S.S.S.); the total nitrogen was analyzed with the variant of Bouat and Crouzet (1965); and the active carbonate (calcium carbonate extractable with ammonium oxalate) was determined according to AFNOR (1982). The exchangeable cations of Ca2+ and Mg2+ were determined using AAS (Philips UP9100x), and K+ and Na+ with a flame photometer (Sherwood 410).

Results

The 18 seed plants of Quercus ilex subsp. ballota showed that the roots had between 40 to 60% of their tips mycorrhized with T. pseudoexcavatum, T. indicum, and T. melanosporum, respectively, without contaminating ectomycorrhizae of other fungi. Tables 1 and 2 show the morphological characteristics of these ectomycorrhizae, to assist in their rapid identification in the routine control procedures for mycorrhized plants in truffle culture.

Table 1 Morphological characters of Tuber melanosporum, T. indicum and T. pseudoecavatum ectomycorrhizae
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Table 2 Anatomical characters of Tuber melanosporum, Tuber indicum and Tuber pseudoecavatum ectomycorrhizae
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The ectomycorrhizae of T. pseudoexcavatum and T. indicum have a considerable morphological similarity with the ectomycorrhizae of T. melanosporum, due to the presence of cystidia with right angle-like ramifications and outer mantle pseudocells with a sinuous puzzle-like form, which up to now have been considered the most distinctive characteristics of T. melanosporum ectomycorrhizae (Palenzona 1969; Zambonelli et al. 1993; Granetti 1995; Granetti et al. 2005). However, some morphological characteristics of T. pseudoexcavatum ectomycorrhizae make it possible to differentiate this species from T. indicum, and T. melanosporum. Specifically, the length of the unramified ends of the ectomycorrhizae in T. pseudoexcavatum is much greater than in the other Chinese truffles ectomycorrhizae, and the T. pseudoexcavatum ectomycorrhizae are darker in color than T. indicum and T. melanosporum ectomycorrhizae. Moreover, the outer mantle pseudocells and cystidia diameter of T. pseudoexcavatum are larger than the pseudocells and cystidia of T. indicum and T. melanosporum (Tables 1 and 2; Figs. 1 and 2).

Fig. 1
figure 1

a Macroscopic appearance of T. pseudoexcavatum mycorrhizae (50×) (bar = 1000 μm); b detail of T. pseudoexcavatum cystidia with right angle-like ramifications (630×; bar = 10 μm); c detail of T. indicum cystidia with right angle-like ramifications (630×; bar = 10 μm); d detail of T. melanosporum cystidia with right angle-like ramifications (630×; bar = 10 μm)

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Fig. 2
figure 2

a Macroscopic appearance of T. indicum mycorrhizae (50×; bar = 2,000 μm); b hyphal pseudocells of the outer surface of the T. indicum mantle with a sinuous form (630×; bar = 10 μm); c hyphal pseudocells of the outer surface of the T. melanosporum mantle with a sinuous form (630×; bar = 10 μm); d hyphal pseudocells of the outer surface of the T. pseudoexcavatum mantle with a sinuous form (630×; bar = 10 μm)

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We obtained the T. pseudoexcavatum and T. indicum ectomycorrhizae using two soil samples with the following properties (Table 3): a relative abundance of sand and clay and moderate level of silt, which generates a texture tending towards sandy clay loam. These soils have a moderately basic pH, low levels of total carbonates but an elevated level of active carbonate. Levels of organic carbon and total nitrogen are moderate and the C/N ratio is 13 to 15. They have high values of exchangeable cation complex and the degree of saturation of exchangeable cations is 100%, with a good proportion of exchangeable Ca2+ and Mg2+, K+ in relatively large concentrations, and scarce Na+. These soils have a good structure with a granular tendency and an abundance of pores.

Table 3 Analytical results of the two soil samples from Tuber melanosporum burns (C.E.C. cation exchange capacity, %V exchangeable cation saturation percentage; Sand, clay, silt, total carbonate, active carbonate, T.O.C., and N expressed in g kg−1; C.E.C. and exchangeable cations expressed in cmol kg−1)
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Discussion

Wang and Hall (2001) confirm that, in Europe, some of the Chinese truffles that were similar to T. melanosporum were originally identified as T. indicum. However, these authors and Wang and He (2002) indicated that most of the truffles that were being exported from southwest China to Europe were T. sinense Tao and Liu and T. pseudoexcavatum, in hundreds of tons a year. T. pseudoexcavatum has also been detected in Japanese truffle markets (Yamanaka et al. 2001).

Our results show that the morphological characteristics of T. pseudoexcavatum ectomycorrhizae enable this species to be differentiated from T. indicum ectomycorrhizae. We also show that the ectomycorrhizae of T. pseudoexcavatum have a similar morphology to the ectomycorrhizae of T. melanosporum, although there are some morphological characteristics which make it possible to differentiate both species. In any case, it is necessary to use molecular tools to positively separate these species. It would, therefore, be very useful to have a specific primer for T. pseudoexcavatum to be used in conjunction with the other black truffle specific primers already designed by Paolocci et al. (1999) for use in multiplex PCRs.

From the soil point of view, very few studies have been done so far on Chinese truffles. Most Chinese truffles have been found in calcareous soils. Tuber furfuraceum Hu and Y. Wang and T. pseudoexcavatum live in calcareous soils (Wang and Li 1991; Riousset et al. 2001; Wang and Hall 2001; Hu and Wang 2005). Tuber sinense carpophores have been found in the 0–30 cm layers of calcareous, high pH, and clay soils (Zhang and Wang 1990; Wang and Hall 2001). T. indicum var. yunnanense Yamanaka carpophores have been found at a depth of 2–12 cm, also in very poor, calcareous, and purple soils, with a pH of 6.5–7.4 (Yamanaka et al. 2000, 2001). Tuber formosanum Hu is found only in calcareous soils with a wide range of soil parameters (pH, total nitrogen, carbon, sulfur, and available nutrients), suggesting that T. formosanum can adapt to a wide range of soil conditions (Hu et al. 2005).

Riousset et al. (2001) report that T. indicum develops at a depth of 30–40 cm under a mulch of pine needles in mountain pinewoods, in soils free from calcium carbonate and with a moderate pH, rich in organic matter and with a high C/N ratio. However, other authors indicate that T. indicum inhabits calcareous soils. Fourré et al. (1996) report that T. indicum occurs on calcareous plateaus at 2,000–3,000 m. Granetti et al. (2005) indicate that T. indicum inhabits calcareous substrates with a pH which varies between 5.5 (due to organic matter) and 8.5.

Our results confirm that T. pseudoexcavatum and T. indicum ectomycorrhizae develop well in calcareous substrates which are rich in active carbonate, whose properties fulfill the necessary values for the development of T. melanosporum according to Callot (1999), Ricard (2003), and García-Montero et al. (2006) (Table 3). Active carbonate is a finely divided fraction of calcareous rock, smaller than 50 μm in size, very chemically active, and constitutes an important reserve of exchangeable Ca2+. The presence of both soil components is very important to T. melanosporum due to the action of several factors (García-Montero et al. 2007a, b). These results contrast with the soils described by Riousset et al. (2001) for T. indicum; these are devoid of calcium carbonate, have a moderate pH and are rich in organic matter. Therefore, these results and the studies of Wang (2006a) have confirmed that T. indicum appears to adapt to a wide range of soil conditions.

Regarding host plants, some authors have reported that T. pseudoexcavatum is associated with pines, whereas T. indicum is associated with pines and Asian Quercus (Cooke and Masse 1892; Zhang and Minter 1988; Riousset et al. 2001; Wang and Hall 2001; Granetti et al. 2005). We have established that T. pseudoexcavatum and T. indicum showed a high capacity for mycorrhization with Quercus ilex subsp. ballota. Comadini and Pacioni (1997), Zambonelli et al. (1997) and Di Massimo et al. (1998) also mycorrhized T. indicum with Quercus ilex L. subsp. ilex, Q. pubescens Willd., and Q. cerris L. These four Quercus taxa are the principal host plants of T. melanosporum in European Mediterranean areas. Ferrara and Palenzona (2001) also indicate that the spores of T. indicum have a high germination capacity, and form young ectomycorrhizae and abundant preotrophic mycelia 3 months after inoculation of spores in Quercus pubescens plants. In this regard, not only T. indicum but also T. melanosporum is able to form mycorrhizae as soon as 2.5 months after inoculation.

We conclude that T. pseudoexcavatum and T. indicum ectomycorrhizae grow well in calcareous substrates rich in active carbonate inhabited by T. melanosporum; they also mycorrhize with the principal T. melanosporum host plants. Therefore, both Chinese truffle species have the potential capacity to penetrate numerous T. melanosporum plantations and Mediterranean ecosystems. The ectomycorrhizae of these Chinese truffles are morphologically similar to T. melanosporum ectomycorrhizae, and it is, therefore, important to develop new protocols, based on morphological and genetic analyzes, for the quality control of commercial plants mycorrhized with Tuber.