Correspondence: Milan Gryndler, Institute of Microbiology, Academy of Sciences of th Czech Republic, Vídeňská 1083, 142 20 Praha 4, Czech Republic. Tel./fax: 00420 41062384; e-mail: firstname.lastname@example.org
The terms ‘brûlé’ and ‘burnt’ are used to describe vegetation-devoid areas of the ground around a range of woody plants interacting with certain truffle species. Increasing interest is currently focused on a systematic search for and study of volatile organic compounds (VOCs) emitted by truffles in the course of their life cycle. These metabolites are now recognized as biochemicals with an important impact on burnt formation. Based on current molecular approaches, Tuber melanosporum is emerging as an aggressive colonizer of the brûlé, dominant in competition with indigenous brûlé-associated organisms, suppressing their richness and biodiversity. There is compelling evidence that mycelia, mycorrhizae, and fruiting bodies of brûlé-forming truffles have evolved diffusible metabolites for their survival, typically characterized as having harmful effects on weeds, impairing seed germination, altering root morphogenesis and plant hormonal balance, or inhibiting the native rhizospheric microflora regularly associated with the brûlé. These effects can be widely interpreted as allelopathic phenomena, and the brûlé may thus be regarded as a promising opportunity to study truffle allelopathy. Considering the outstanding success of the genome analysis in T. melanosporum, we are facing a very difficult task to proceed from the molecular to the ecological level.
Circular zones with scanty vegetation around host plants colonized by some ectomycorrhizal fungi have been known for a long time (Ciccarello, 1564). This phenomenon commonly referred to by the French term brûlé (Fig. 1) is based mainly on phytotoxic effects of metabolites emitted by some Tuber species, typically Tuber melanosporum, Tuber aestivum, and Tuber indicum, which affect the herbaceous cover and roots of host plants in this particular ecosystem (Pappa, 1980; Pacioni, 1991; Plattner & Hall, 1995; Lanza et al., 2004). The denuded area appears around a range of deciduous woody plants, including oak (Quercus spp.), hazel (Corylus spp.), poplar (Populus spp.), beech (Fagus spp.), and cistus (Cistus spp.) Occurrence of brûlés with other truffle species is controversial (Hall et al., 2009). On the other hand, among the few plants which are able to grow in the brûlé, Sedum altissimum, Festuca ovina, and Hieracium pilosella are indicative of the truffle presence (Delmas, 1983; Sourzat, 1993; Plattner & Hall, 1995; Splivallo, 2007). Although appearance of a brûlé is a sign that the above truffle species are active in the ground, it does not secure the production of ascocarps. Each year the host plant begins the season with root elongation and ectomycorrhization of their tips and closes the season by dormancy of roots and death of old mycorrhizae. As a consequence, brûlés continue to grow larger each year. The first distinct brûlé appears under host woods and shrubs in < 2 years and may continue to enlarge up to 10–15 years, with extension of the affected area to 15–20 m. Host plants with quicker growth produce brûlé faster.
The aim of the present minireview is to summarize the current state of knowledge on the truffle strategy based on volatile secondary metabolites emitted in the course of their life cycle. These metabolites are now recognized as biochemicals with important impact on brûlé formation. Although genes responsible for the brûlé appearance have not yet been identified, recent discoveries (Martin et al., 2010; Martin, 2011) move truffle research into the field of ecosystem science.
Truffle life cycle
The vegetative stage of T. melanosporum is represented by a primary free-living uninuclear mycelium (Paolocci et al., 2006) derived from spores. This mycelium is a facultative transitory saprotroph that obtains nutrients by decomposing dead and decaying organic matter from the soil. It is speculated that the saprotrophic ability of the free-living mycelium is low and its survival very limited and intended to give rise to mutualistic symbiotic associations in contact with roots of a wide range of host plants (Martin et al., 2010; Martin, 2011; Rubini et al., 2011a).
The switch to the symbiotic stage is manifested by the formation of ectomycorrhizae that consist of hyphae intimately associated with the intercellular space of the peripheral tissue of apical root tips. Both partners exchange commodities required for their proper growth and survival. Carbon compounds of photosynthetic origin from the host plant are directed to the fungus, and various nutrients, especially inorganic nitrogen and phosphate, are transported from the fungus to the symbiotic plant. In addition, the fungus helps the host tree to support high calcium levels and offers a source of moisture (e.g. Smith & Read, 2008). In the light of new molecular data, the mutualistic exchange of nutrients was shown to involve specific genes/proteins in combination with environmental factors, such as nutrient shortage (Martin et al., 2001; Martin & Nehls, 2009). Recently, increased fluxes of carbohydrates, oligopeptides, amino acids, and polyamines were identified at the symbiotic interface of both partners (Martin et al., 2010).
For a long time, T. melanosporum was thought to be homothallic or exclusively selfing (Bertauld et al., 1998). To date, molecular investigations have brought a profound reevaluation of the sexuality of T. melanosporum having also implication for other truffle species. The current concept envisages a haploid extraradical mycelium composed of hyphae of opposite sexual polarity, which are non-uniformly distributed in their natural localities, and after obligate outcrossing (Martin et al., 2010; Rubini et al., 2011a, b), they give rise to subterranean hypogeous ascocarps. It is envisaged that control of outcrossing by mating (MAT) genes and most sex- and fruiting-related pathways identified in T. melanosporums is comparable with other heterothallic ascomycetes (Paolocci et al., 2006; Rubini et al., 2007, 2011a, b; Murat & Martin, 2008a; Ricionni et al., 2008; Belfiori et al., 2009; Napoli et al., 2010).
Bioactive truffle VOCs
In addition to research on metabolites involved in nutritional exchange between the fungus and the host plant, increasing interest is currently focused on a systematic search and study of volatile organic compounds (VOCs) emitted by truffles. Their phytotoxic effect was first noted in the early nineties (Pacioni, 1991).
To date, around 200 VOCs from various truffle species have been identified, some of them described for the first time (Splivallo, 2007). Their number is likely to continue growing (Tarkka & Piechulla, 2007). They are generally simple aliphatics containing functional groups such as alcohols, aldehydes, aromatic compounds, esters, furans, hydrocarbons, ketones, and nitrogen- and sulfur-containing compounds (Splivallo et al., 2007a). Some are common to all truffle species studied so far, and others are species-specific. A list of truffle secondary metabolites reported in the literature can be found in Splivallo (2008). Examples of some truffle VOCs structures are shown in Fig. 2. A recent exhaustive current survey of the field is presented in Splivallo et al. (2011).
VOCs are released in the course of the entire truffle life cycle, including free-living mycelia, mycorrhizae, and ascocarps (Talou et al., 1989; Menotta et al., 2004; Zeppa et al., 2004; Splivallo et al., 2007a, 2009, 2011). Profound metabolic differences between vegetative growth and the fructification stage are documented by aromatic spectra that differ in free-living mycelia versus ascocarps (Splivallo et al., 2007a).
The first biological activity of truffle volatiles was demonstrated in vitro in aqueous extracts of ascocarps of T. melanosporum, Tuber borchii, and T. indicum tested on host Cistus incanus and non-host Arabidopsis thaliana plants. Ten volatile compounds of the natural truffle aroma – 3-methyl-1-butanol, 1-hexanol, 1-octen-3-ol, 3-octanol, 3-octanone, trans-2-octenal, 2-phenylethanol, dimethyl sulfide, 3-methylsulfanylpropanol, and 2,3-butanediol – were tested. Volatiles from ascocarp extracts as well as their corresponding synthetic VOCs showed distinct phytotoxic effects manifested by strong inhibition of roots and cotyledon leafs and bleaching of seedlings, indicative of modification of the oxidative metabolism of test plants. The strongest phytotoxic effect on host plants was obtained with 1-octen-3-ol and trans-2-octenal (Splivallo et al., 2007b).
A breakthrough in the field occurred by analysis of mycelial activities of the three above-mentioned truffle species grown in submerged liquid culture (Splivallo et al., 2009). In dual cultures of mycelium/plant, mycelia were found to exude the diffusible fungal phytohormones auxin (indole-3-acetic acid, IAA) and ethylene which, depending of the Tuber species, affected root morphology in the course of the premycorrhizal stage without direct physical contact of emitting mycelia with both host C. incanus and non-host A. thaliana plants. Although limited to in vitro observations, this was a confirmation that truffles are capable of sending specific signals over a distance. In nature, both auxin and ethylene act presumably jointly as underground inhibitory signals on host and non-host plants, rather than as early mycorrhization signals, as supposed by Menotta et al. (2004). This view is supported by the agronomical practice of applying comparatively high doses of both auxin and ethylene as potent herbicides for the management of dicot weeds (Grossmann, 2003). The fact that both host and non-host plants were inhibited suggests a complex and widespread inhibitory action range of the above phytohormones likely to be active in brûlé development and/or maintenance. Nevertheless, confirmation of this role awaits quantification of both ethylene and indole-3-acetic acid in truffle-ground soils.
Phenolic truffle VOCs
VOCs with aromatic rings, having a typical phenolic odor, are another rich source of toxic phytochemicals present in the brûlé. Earlier studies have already revealed differences in chemical composition of soils inside and outside the brûlé (Montacchini & Caramielle Lomango, 1977). Correspondingly, soil samples taken from the brûlé generally have higher polyphenol content than those collected from outside the brûlé (Splivallo, 2007). This paragraph focuses exclusively on phenolic VOCs related to truffles, without considering soil phenolics produced by other microorganisms, host plants, and litter decomposition. Their biological importance was demonstrated by Gryndler et al. (2009).
The coumarins – scopoletin, angelicin, and bergapten, were reported in ascocarps of some Tuber species, and their mutual pattern was proposed as a distinguishing chemotaxonomic marker (Tirillini & Stoppini, 1996). Coumarin derivatives with phytotoxic effects (e.g. bergapten) were also identified in methanolic truffle extracts (Tirillini & Granetti, 1995; Tirillini & Stoppini, 1996; Angelini et al., 2009). According to Splivallo (2007), individual phenolic compounds were detected in low concentration in various Tuber species, but their role in truffle metabolism awaits further study. Recently, 17 aroma active compounds of the T. melanosporum scent were identified and some of them were reported as particularly rich in phenols. Among newly identified aroma molecules, T. melanosporum was found to emit mostly 3-ethyl-5-methylphenol (quantitatively more than 50% of the aroma molecules emitted), 5-methyl-2-propylphenol, ß-phenylethanol, and 3-ethylphenol, whereas T. aestivum was estimated to emit up to 100 times less aroma compounds, mainly ß-phenylethanol, dimethyl sulfide, and 3-methylphenol (Culleré et al., 2010).
In fact, phenolic compounds are the most frequently reported groups of allelochemicals, widely distributed in the soil environment (Einhellig, 1995). We found in field experiments that the T. melanosporum brûlé periphery showed an increased content of phenolic 1,2,4-substituted trihydroxybenzene structure, as compared with the surrounding soil and the central area of the brûlé (Fig. 3). The higher content of the phenolics corresponds with the circular aggressive zone of the brûlé periphery (Sourzat, 2004; Chevalier 2010). Our finding provides an opportunity for the study of presumed allelopathic interactions operative within the brûlé.
We can assume that during evolution, truffles have evolved a dispersal system relying on a strong scent that enables animals and some insects to locate typically belowground truffle fruitbodies (Trappe & Claridge, 2010). In this context, complementary roles of VOCs in truffle dissemination have emerged. Truffles suppress the vegetation within the brûlé to mark out its location in the terrain, and at the same time, they emit volatile signals to attract free-living organisms (e.g. wild boar, deer, bears, mice, rabbits, insects) acting as vectors for truffle dispersal.
Traditionally, domestic pigs and trained dogs are used by truffle hunters to locate belowground truffle fruitbodies in nature. The strong musky scent of T. melanosporum contains 5-α-androstenol, a steroidal hormone close to human androsterone, which has been identified in salivary glands of boars (Claus et al., 1981). Interestingly, this steroid alone is ignored by pigs and trained dogs. Whether this compound is perceived by free-living mammals in nature has not been tested. On the other hand, pigs and trained dogs are attracted by dimethyl sulfide, the key compound of the smell of T. melanosporum identified by Talou et al. (1990).
Chemical structures and important biosynthetic pathways of a number of volatile-based constituents of the T. melanosporum aroma have been clarified at the molecular level only recently. Micro-extraction analysis of individual volatile sulfur compounds from ascocarps confirmed the presence of a mixture of different classes of volatiles containing functional groups of alcohols, aldehydes, ketones, acids, esters, amines, aromatic ethers, hydrocarbons, and sulfur compounds (Pelusio et al., 1995). At the genetic level, the pioneering work of Martin et al. (2010) revealed a 125-megabase T. melanosporum haploid genome as one of the largest and most complex fungal genomes sequenced to date, containing only about 7500 protein-coding genes and about 58% mobile genetic elements. The genome-derived sequence data suggest that the fungus possesses most, if not all, genes needed to synthesize the key constituents of the truffle aroma. A large set of VOC-related genes plus genes involved in sulfur metabolism and amino acid degradation pathways were found to be particularly expressed in T. melanosporum ascocarps. Current truffle research focuses on the identification and description of key biosynthetic routes operating in truffles with special emphasis on the understanding of the genetic background of enzymes and pathways involved in biosynthesis of the truffle aroma (Splivallo et al., 2011).
It turns out that the final aroma of T. melanosporum ascocarps depends upon the endogenous enzyme complement of the fungus that may differ in various geographical regions (Gioacchini et al., 2008), rather than upon metabolic activities of bacteria (Barbieri et al., 2005) and yeast (Buzzini et al., 2005) living in association with truffle fruitbodies in distinct geographical locations, as envisaged earlier by many truffle researchers. However, whether volatiles produced by accompanying microorganisms may contribute to the final truffle scent is not yet clear.
Until recently, only cultivable soil fungi were identified and studied in natural truffle grounds at the traditional population/community resolution level (Luppi-Mosca, 1972; Luppi & Fontana, 1977; Bedini et al., 1999; Citterio et al., 2001; Sbrana et al., 2002) without real insight into interactions of microbial communities coexisting in the brûlé. Thanks to current molecular approaches applied to truffle-ground soils, it was recently shown that terrestrial fungal community composition and its temporal dynamics are fundamentally different inside and outside of the brûlé and change successively in the course of brûlé development (Napoli et al., 2008, 2010). Based on these findings, T. melanosporum is emerging as an aggressive invasive colonizer of the brûlé, dominant in competition with other brûlé-associated organisms and suppressing their richness and biodiversity (Martin et al., 2010; Napoli et al., 2010). Additionally, a dynamic temporal succession of individual Tuber species was recorded during brûlé development in truffle fields (Serra et al., 2007). In general, T. melanosporum seems to be able to fully eliminate competing indigenous ectomycorrhizal fungi, including Tuber species, present as contamination of T. melanosporum brûlés (García-Montero et al., 2008; Zampieri et al., 2009; Napoli et al., 2010). It should be added that the native T. melanosporum is threatened by a closely related, similarly-looking but less aromatic Chinese truffle (T. indicum) showing a superior competitiveness compared with the locally grown Périgord black truffle (Murat et al., 2008b).
VOCs emitted by truffles belong generally to secondary metabolites that are not required for their basic life processes such as growth, development, and reproduction, but are the source of multiple volatile-based interactions with other organisms living inside the truffle ground.
There is compelling evidence that presymbiotic truffle mycelia (Tirillini et al., 2000; Menotta et al., 2004; Splivallo et al., 2007a, 2009), mycorrhizae (Menotta et al., 2004), and ascocarps (Mauriello et al., 2004; Zeppa et al., 2004; Splivallo et al., 2007a; Culleré et al., 2010) have evolved diffusible metabolites as important tools for their survival, typically characterized as having harmful effects on weeds, impairing seed germination, causing necrosis of roots of host and non-host plants and altering host-plant hormonal balance or inhibiting the native rhizospheric microflora regularly associated with the brûlé (Fasolo-Bonfante et al., 1971; Pacioni, 1991; Plattner & Hall, 1995; Lanza et al., 2004; Zeppa et al., 2004; Splivallo, 2007, 2008; Splivallo et al., 2007b, 2009; Napoli et al., 2010). Systematic investigations of brûlé VOCs in vivo will start with the construction of a ‘volatile map’ of the truffle aroma in the field (Bohannon, 2009).
Brûlé has been hypothesized to be caused by competition of the truffle mycelium for nutrients and/or water (Delmas, 1983). Alternatively, the brûlé could be explained by parasitism, manifested by severe necrosis of root cortices in brûlé-located weeds (Plattner & Hall, 1995; Chevalier, 2010) or by abscission and scar formation on roots of host trees (Sourzat, 2004). Finally, the brûlé development could be assigned to phytotoxic substances released into the brûlé environment (Fasolo-Bonfante et al., 1971; Pacioni, 1991; Lanza et al., 2004).
Traditionally, the brûlé is considered as a marker of the truffle presence. Its development is explained by the virulence (or rather aggressiveness) of the fungus vis-à-vis the environment and defined by mutual relations between the radius of brûlés and fitness of host trees. It has been shown that the expansion of the brûlé brings about conspicuous changes in the root system of the host with location of the most aggressive competition zone at the brûlé periphery (Sourzat, 2004, 2008, 2010; Chevalier, 2007, 2010).
There is no doubt that the harmful processes listed previously do occur in the brûlé but do not seem adequate to fully explain the striking burnt-associated harm observed in nature.
Revolutionary insight into the truffle ecosystem dynamics based on a set of metagenomic techniques (Napoli et al., 2008, 2010; Mello et al., 2010, 2011) suggests, but does not prove, that allelopathy may be operative in the brûlé. It should be mentioned that the allelopathic potential of truffles has already been taken into consideration by a number of researchers (Tirillini & Granetti, 1995; Tirillini & Stoppini, 1996; Chevalier, 1998; Olivier et al., 2002; Splivallo, 2007; Angelini et al., 2009).
In simple terms, allelopathic inhibition is commonly defined as introduction of harmful biomolecules (allelochemicals) by an organism into the environment to induce a number of interacting processes in the receiving species (plant, fungus or microorganism) present in this environment (Rice, 1984). Once the allelochemical enters this environment, additional bioregulatory and abiotic interactions may take place (Einhellig, 1995; Pedrol, 2006). Allelopathy may thus offer possible explanation for the aggressiveness of T. melanosporum and for strong cytotoxic effects manifested in the brûlé.
The best way to study allelopathy is to find signs of it occurring in nature. The distinct truffle brûlé is one of the rare exceptions enabling direct observation of the presumed allelopathy symptoms in nature. Based on current knowledge of brûlé behavior, truffles seem to have established an efficient environmental strategy to gain and control the space needed for their survival and reproduction. Additionally, the truffle brûlé may be regarded as a very promising field to study allelopathy at the general level.
Although knowledge of truffle volatile biochemicals in vitro is expanding, it is not yet connected to parallel studies in vivo in truffle-ground soils. Monitoring effects of truffle biochemicals in nature is far more complex than in artificial conditions, and their bioavailability and effective concentrations must be checked in combination with other biotic (host and non-host plants, competing fungi, insects, soil bacteria) and abiotic factors (e.g. temperature, soil composition and structure, pH, drainage, aeration, humidity) which may be of prime importance to the brûlé ecology.
It is expected that the emerging field of ecogenomics coupled with chemical ecology will further the knowledge of the mode of action of truffle biochemicals and their roles in the efficient life strategy of the fungus. Understanding of how truffles function and interact in the ecosystem is vital for the future progress in the field (Martin, 2011). Considering the outstanding success of the genome analysis in T. melanosporum (Martin et al., 2010), we are facing a very difficult task to proceed from the molecular to the ecological level. This research offers considerable challenges.
This work was supported by the grant P504/10/0382 of the Czech Science Foundation and by the Institutional Research Concept AV0Z50200510 of the Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague. We would like to thank Ján Gažo and Marián Miko from the Slovak University of Agriculture in Nitra for critically reading the manuscript.