Introduction

Boletus Fr. is a cosmopolitan genus of ectomycorrhizal fungi widely represented in the temperate zones of Northern and Southern Hemispheres. The genus comprises more than 1,000 species with epigeous fructification, inhabiting forests in tropical and mid latitudes. Boletus forms ectomycorrhizas with a large number of suitable hosts: Fagales-Fagaceae (Castanea, Castanopsis, Fagus, Lithocarpus, Quercus) and Betulaceae (Carpinus, Corylus, Betula, Ostrya, Populus); Malvales-Malvaceae (Tilia) and Cistaceae (Cistus); Malpighiales-Salicaceae (Salix); Ericales-Ericaceae (Arctostaphylos); and Pinales-Pinaceae (Abies, Keteleeria, Picea, Pinus, Tsuga) (Olivier et al. 1997; Águeda et al. 2006; Mello et al. 2006).

The Boletus edulis species complex (B. edulis Bull. sensu stricto, Boletus aereus Bull., Boletus pinophilus Pilát & Dermek, and Boletus reticulatus Schaeff.) has great economic importance for its edibility (Singer 1986; Hall et al. 1998) being B. edulis a major commercial mushroom consumed worldwide. This fungal species is collected exclusively from the wild (Cannon and Kirk 2007) and no controlled production has been done to date.

Edible mycorrhizal mushrooms are not only a gourmet food but also a source of income for collectors (Wang and Hall 2004). Total annual worldwide consumption of B. edulis complex is between 20,000 and 100,000 tons (Hall et al. 1998). Important markets include North America, France, Italy, and Germany (Hall et al. 1998). The estimated annual production of B. edulis species complex from the autonomous community of Castilla y León in Spain, is 8,500 tons, worth approximately 38 million Euros (Martínez-Peña et al. 2006-2008).

In some regions of Central Spain (Zamora, León, and Salamanca provinces) with abundant fires and dominated exclusively by Cistus ladanifer, B. edulis sporocarps are regularly observed. Águeda et al. (2006) described field ectomycorrhizas formed by these organisms and sequenced them for taxonomic verification. This fact is important since Cistus species occur in degraded areas where economic resources are scarce to maintain human population. The family Cistaceae, with eight genera and almost 200 species (Muñoz and Navarro 1993) is distributed primarily in the temperate areas of Europe and the Mediterranean basin, but is also found in North and South America (López González 2001). The genus Cistus is represented in the Iberian Peninsula by 12 shrub species, all occurring during primary succession of tree stands. Cistaceae species are pyrophytic in general, and their germination is benefited by high temperatures where they are adapted to fires in Mediterranean forests (Alonso et al. 1992). Cistus species often form pure stands in vast areas heavily subjected to fire and/or grazing because of their ability as early colonizers after disturbance.

Cistus can form both ecto- and arbuscular mycorrhizas (Smith and Read 1997). More than 200 fungal ectomycorrhizal species belonging to 40 genera are reported to associate with Cistus (Puppi and Tartaglini 1991; Comandini et al. 2006). Rockroses (Cistus and Helianthemum sp.) are ecologically important species because they may act as a reservoir of mycorrhizal fungi after a forest disturbance (Torres et al. 1995; Díez 1998).

Among the edible mycorrhizal mushrooms, only Tuber melanosporum Vittad. and Tuber uncinatum Chatin have been cultivated commercially (Wang and Hall 2004).

Also, some success has been achieved with Lactarius deliciosus (L.) Gray, Lyophyllum shimeji (Kawam.) Hongo, Tuber borchii Vittad., and Rhizopogon roseolus (Corda) Th. Fr. (Wang and Hall 2004). Few attempts to produce edible sporocarps using a Cistaceae host have been reported, most of them involving T. melanosporum and Cistus sp. (Chevalier et al. 1975; Díez et al. 1994; Fontana and Giovanetti 1978; Giovannetti and Fontana 1982; Roth-Bejerano et al. 2003; Wenkart et al. 2001), or Terfezia claveryi Chatin and Helianthemum sp (Morte et al. 2004).

Ectomycorrhizal synthesis experiments are useful to determine fungus–plant host compatibility and for morphological and physiological research (Giomaro et al. 2005). In this paper, this technique has been used to test the ability of the B. edulis species complex to form ectomycorrhizas with Cistus sp. under controlled conditions as well as provide detailed anatomical descriptions of the formed ectomycorrhizas. This research is part of a project aimed at promoting shrub inoculations for the production of edible mycorrhizal fungi (Martínez-Peña et al. 2007).

Materials and methods

Fungal isolates

Fungal isolates of Boletus aereus, B. edulis, B. reticulatus, and B. pinophilus were obtained from sporocarps collected in northern Spain (Table 1). Isolations were made by explants from sporocarp tissues plated on modified Melin–Norkrans agar culture medium (Marx 1969) or biotin-aneurin-folic acid agar culture medium (BAF) (Oort 1981) and maintained by transferring to fresh media every 3 months.

Table 1 Fungal isolates used in the ectomycorrhizal synthesis
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The identification of the fungal species was confirmed by molecular analysis of the internal transcribed spacer (ITS) of the nuclear rDNA region (Leonardi et al. 2005).

Amplicons obtained with the ITS1/ITS4B primers (Gardes and Bruns 1993) from each fungal isolate were purified and sequenced in both directions. The ITS sequences obtained were compared with sequences deposited in GenBank to confirm their taxonomic identification, and submitted to the GenBank databases under accession numbers reported in Table 1.

Pure culture synthesis procedures

Cistus albidus L. and C. ladanifer L. seeds were rinsed in 90°C water for 30 min, maintained in 30% sodium hypochlorite for 10 min, and washed in sterile distilled water. Disinfected seeds were placed on BAF agar Petri dishes and stratified for 2–3 weeks at 4°C. Plates were then placed at room temperature for seed germination (20–23°C). After 2 weeks of incubation, seeds showing contamination were discarded.

Aseptically germinated seedlings (radicle 1–2 cm long) were transferred into ectomycorrhizas synthesis tubes (Molina 1979) filled with a sterilized mixture of 10 ml peat, 110 ml vermiculite and 60 ml BAF nutrient solution modified reducing the glucose to 20 g/l. The synthesis tubes were inoculated with 10 ml of a mycelium culture of either B. aereus, B. edulis, B. reticulatus, and B. pinophilus grown in BAF liquid medium. All the fungi were tested with C. albidus and C. ladanifer with four replicates for each fungus–host combination. Synthesis tubes with the inoculated seedlings were grown for 4–5 months at 20–25°C under fluorescent lights (150 μmol s-1 m-2 [400–700 nm], 16 h/day). At the end of the growing period, seedlings were removed from the synthesis tubes and root systems washed and examined for ectomycorrhizal formation.

Morphological description of ectomycorrhizas

Ectomycorrhizal roots and rhizomorphs were carefully extracted with the aid of a stereomicroscope, fixed in FAA (Agerer 1986) and stored as voucher specimens in the Dpto. Inv. Exp. For. Valonsadero (Soria, Spain). The general methodology and terminology for characterizing the ectomycorrhizas follows Agerer (1987-2006, 1991) and Agerer and Rambold (2004-2008). For the observation of the mantle, the ectomycorrhizas were grated with the peeling technique (Agerer 1991). Mantle and rhizomorph preparations of fresh ectomycorrhizas were fixed on slides with lactic acid for microscope observation.

Results

B. aereus, B. edulis, and B. reticulatus formed ectomycorrhizas with C. ladanifer and C. albidus, whereas B. pinophilus did not form ectomycorrhizas with either Cistus host (Table 2).

Table 2 Formation of ectomycorrhizas in each host–fungus combination
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Mycorrhizal structures of each fungal species were identical in both Cistus species as it has been described for other host–fungus combinations (Agerer and Rambold 2004-2008). Consequently, only one complete description is given for each fungal species indicating the host plant taken into account in each case. Herbarium codes for the described mycorrhizas were: VALONSADERO-MYCORRHIZA 049 for B. aereus and C. ladanifer (from strain 393 IRTA), VALONSADERO-MYCORRHIZA 047 for B. edulis and C. albidus (from strain 375 IRTA), and VALONSADERO-MYCORRHIZA 045 for B. reticulatus and C. albidus ectomycorrhizas (from strain 1054 valonsadero).

B. aereus + C. ladanifer

Mantle type: plectenchymatous, colorless, clamps lacking, outer mantle layer with ring-like arrangement of hyphal bundles, Type A (Fig. 1b); middle mantle layer hyphae arrangement without pattern; inner mantle layer hyphae arrangement with broad streaks of parallel hyphae. Tip with the same structural characteristics as in the older parts of mantle.

Fig. 1
figure 1

Morphological and anatomical characters of the ectomycorrhizas obtained in pure culture synthesis. a Ectomycorrhizas and rhizomorphs of Boletus aereus and Cistus ladanifer. Bar = 10 mm. b Outer mantle layer of Boletus aereus and Cistus ladanifer. Bar = 10 µm. c Surface of rhizomorph with cystidia of Boletus aereus and Cistus ladanifer. Bar = 10 µm. d Ectomycorrhizas and rhizomorphs of Boletus edulis and Cistus albidus. Bar = 10 mm. e Outer mantle layer of Boletus edulis and Cistus albidus. Bar = 10 µm. f Rhizomorph with vessel-like hyphae of Boletus edulis and Cistus albidus. Bar = 10 µm. g Ectomycorrhizas and rhizomorphs of Boletus reticulatus and Cistus albidus. Bar = 10 mm. h Middle mantle layer of Boletus reticulatus and Cistus albidus. Bar = 10 µm. i Surface of rhizomorph with cystidia of Boletus reticulatus and Cistus albidus. Bars = 10 µm

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Rhizomorphs: up to 15 mm, highly differentiated, boletoid, with vessel-like hyphae with partially or even completely dissolved septa centrally arranged; forming nodia at branching points, clamps absent, colorless, without short inflated cells. Peripheral hyphae similar to mantle cystidia, colorless, smooth (Fig. 1c).

Cystidia: awl-shaped, bristle-like, present on outer mantle layer and on rhizomorphs; ramification presence-position absent or proximal, monopodial or bifurcate; branches ramification absent; septa: present, simple, septa number 2–3; surface smooth.

Emanating hyphae: clamps lacking, straight, colorless; ramification approximately 90°, adjacent to septum, one side-branch at septum; cells even or slightly constricted, smooth.

Exploration type: long distance.

Hydrophobic.

Morphological characters: ectomycorrhizal system solitary, monopodial-pyramidal or irregularly pinnate; unramified ends straight, not inflated, cylindrical, yellowish; mantle surface smooth and glistening, loosely woolly or forming rings (reticulate) (Fig. 1a). Emanating hyphae present, infrequent, not specifically distributed. Rhizomorphs round or roundish, white, frequently ramified at restricted points, connection to mantle kind distinct, origin location proximal, surface smooth or hairy.

B. edulis + C. albidus

Mantle type: plectenchymatous, colorless, clamps lacking; outer mantle layer ring-like arrangement of hyphal bundles, Type A (Fig. 1e); middle mantle layer hyphae arrangement ring-like; inner mantle layer hyphae arrangement with broad streaks of parallel hyphae. Tip with the same structural characteristics as in the older parts of mantle.

Rhizomorphs: highly differentiated, boletoid, with vessel-like hyphae with partially or even completely dissolved septa centrally arranged; forming nodia at branching points, clamps absent, colorless, without short inflated cells (Fig. 1f).

Cystidia: lacking.

Emanating hyphae: clamps lacking, wavy to slightly tortuous, colorless; ramification Y-shaped, adjacent to septum, one side-branch at septum; cells slightly constricted, smooth.

Exploration type: long distance.

Hydrophobic.

Morphological characters: ectomycorrhizal system monopodial-pyramidal or irregularly pinnate, dichotomous-like; unramified ends straight, not inflated, cylindrical, white to yellowish getting more yellow with age; mantle surface shiny and silvery, loosely woolly (Fig. 1d). Emanating hyphae present, abundant, not specifically distributed. Rhizomorphs round or roundish, white, frequently ramified at restricted points, connection to mantle kind distinct, surface smooth or woolly.

B. reticulatus + C. albidus

Mantle type: plectenchymatous, colorless, clamps lacking; outer mantle layer with hyphae rather irregularly arranged with no special pattern discernable (Type B); middle mantle layer hyphae arrangement without pattern or ring-like (Fig. 1h); inner mantle layer hyphae arrangement ring-like or with broad streaks of parallel hyphae. Tip with the same structural characteristics as in the older parts of mantle.

Rhizomorphs: highly differentiated, boletoid, with vessel-like hyphae with partially or even completely dissolved septa centrally arranged; forming nodia at branching points, clamps absent, colorless, without short inflated cells, clamps absent. Peripheral hyphae not specialized or roundish, inflated, as short cells, smooth (Fig. 1i).

Cystidia: two different types, type 1 present on outer mantle layer and on rhizomorphs and type 2 present only on rhizomorphs. Type 1 thin-walled, slightly tapering, often rather similar to ends of normal hyphae, ramification presence-position absent or proximal, monopodial or bifurcate, branches ramification absent, septa present, simple, septa number 1–3, surface smooth. Type 2 globular, ramification presence-position absent, septa absent, cell wall color similar to mantle cells, surface smooth.

Emanating hyphae: clamps lacking, wavy or irregularly inflated or even beaded, colorless; ramification acute or Y-shaped, adjacent to septum, one side-branch at septum; cells even or slightly constricted, smooth.

Exploration type: long distance.

Hydrophobic.

Morphological characters: ectomycorrhizal system: solitary, monopodial-pinnate or coralloid; unramified ends straight or bent, not inflated, cylindrical, white to yellowish getting more yellow with age; mantle surface shiny and smooth, silvery in some zones, loosely grainy or warty (Fig. 1g). Emanating hyphae present, infrequent, not specifically distributed. Rhizomorphs round or roundish, white, frequently ramified at restricted points; connection to mantle kind distinct, surface: smooth.

Discussion

Of the eight tested combinations, only B. pinophilus failed to form ectomycorrhizas with either host, despite the extensive growth of the fungus in the substrate and the adequate root development of both Cistus species. Sporocarps of B. aereus, B. edulis, and B. reticulatus are found associated with Cistaceae plants in the Mediterranean region (Oria de Rueda 2007), while sporocarps of B. pinophilus are associated to Pinaceae or Fagaceae species. The incompatibility of B. pinophilus and Cistus spp. must be confirmed or rejected by testing a wider range of fungal strains.

Over the last 40 years many researchers have synthesized ectomycorrhizas of the B. edulis species complex on various hosts. Froidevaux and Amiet (1975) synthesized B. edulis and Pinus mugo Turra ectomycorrhizas; Tozzi et al. (1980) obtained ectomycorrhizas of B. edulis and Quercus pubescens Willd.; Molina and Trappe (1982a, b) synthesized ectomycorrhizas between B. edulis and eight hosts in the genera Arbutus, Arctostaphylos, Larix, Picea, Pinus, and Tsuga; Ceruti et al. (1983-1984) obtained B. aereus and Q. pubescens ectomycorrhizas; Ceruti et al. (1985) obtained mycorrhizas of B. aereus and Castanea sativa Mill.; Poitou et al. (1982) synthesized ectomycorrhizas in pure culture between Pinus radiata D. Don and B. edulis and B. aereus; and Duñabeitia et al. (1996) obtained B. pinophilus ectomycorrhizas by inoculating P. radiata seedlings with a spore suspension of 106–107 spores per plant.

Seedlings inoculated with Boletus species have been outplanted in attempts to promote fruiting of the valuable edible sporocarps. Olivier et al. (1997) describes plantations of C. sativa and Pinus uncinata Ramond ex DC. inoculated with B. edulis and B. aereus. Meotto et al. (1999) outplanted C. sativa seedlings inoculated with mycelial cultures of B. edulis. Unfortunately, sporocarp production has not been reported from either study.

Description and identification of ectomycorrhizas have evolved greatly following the systematic studies by Agerer (1986, 1987-2006, 1991) and molecular characterization based on DNA analysis (Gardes and Bruns 1993). Although there are some descriptions for Boletus mycorrhizas, most are not precise (De Román et al. 2005). The ectomycorrhizas of the three Boletus species obtained in this study are very similar and fit well with the characters described in Agerer (2006) for this genus: plectenchymatous mantles from the types A, B, or C, boletoid rhizomorphs with nodes and with or without short inflated cells, emanating hyphae smooth or covered by crystals, clamps lacking, cystidia lacking or with cystidia-like hyphal ends, and white to yellowish hydrophobic ectomycorrhizas. All, B. aereus, B. edulis, and B. reticulatus, form white monopodial-pinnate ectomycorrhizas with three-layered plectenchymatous mantle on plan view and boletoid rhizomorphs. B. aereus forms monopodial-pyramidal ectomycorrhizas with ring-like outer mantle layer, and emanating cystidia formed by two or three short cells on the outer mantle layer and rhizomorphs. B. edulis has ring-like outer and middle mantle layers, without cystidia and rhizomorphs also without cystidia or globular cells. B. reticulatus has ring-like or with broad streaks of parallel hyphae inner mantle layer, emanating cystidia on the outer mantle layer and globular and awl-shaped cystidia on rhizomorphs.

The cystidia of B. aereus and B. reticulatus ectomycorrhizas are similar to those present on the hymenia of some Boletus species, like B. reticulatus, B. edulis, or Boletus regius Krombh., appearing as fusiform or globoid structures (Muñoz 2005). However, it can not be discounted that the formation of these elements, as well as the emanating hyphae on B. edulis, could be influenced by the experimental conditions of the synthesis tubes.

The abundance and size of rhizomorphs found in this study, especially for B. aereus, conform to those found in the field. In natural conditions, the mycelium of Boletus is concentrated as rhizomorphs with a high degree of spatial heterogeneity. However, the type of soil could determine the spread of exploratory elements of the Boletus ectomycorrhizas, such as cystidia, rhizomorphs and emanating hyphae, as demonstrated for Lactarius deliciosus mycorrhizal seedlings (Hortal et al. 2008).

There are no previously reported descriptions of B. aereus ectomycorrhizas. All previous descriptions of B. edulis ectomycorrhizas (Ceruti et al. 1987-1988; Garrido 1988; Gronbach 1988; Agerer and Gronbach 1990; Franz and Acker 1995; Palfner 2001; Agerer and Rambold 2004-2008; Águeda et al. 2006) report characters that fit with those we described here. Ceruti et al. (1983-1984), Ceruti et al. (1985), and Garrido (1988) only described the characters of mantle on cross-sections of B. reticulatus, so comparison with our study is not possible.

Considering that fungus is the main factor for determining the anatomical structures of ectomycorrhizas, those are identical for the same fungal species regardless of the host. Although morphological characters are mainly driven by the plant genus, some fungi can control, at least partially, the final form (Agerer and Rambold 2004-2008). Both aspects are true for the three described B. edulis complex ectomycorrhizas, which show the same structures when associated to Cistus as compared to other Pinaceae and angiosperm hosts.

The natural sporocarp production of B. edulis in association with C. ladanifer (Águeda et al. 2006) offers an alternative economic resource for marginal and inland areas with low incomes. Controlled mycorrhization with B. edulis on Cistus and outplanting of inoculated seedlings might be a feasible and promising way to exploit this symbiosis providing economic benefits. To accomplish this, further research is needed to determine the appropriate inoculation methods with compatible Boletus strains, the persistence of Boletus ectomycorrhizas on outplanted, inoculated seedlings, and the factors inducing sporocarp production.