Aromatic weapons: truffles attack plants by the production of volatiles

Truffles, fungal species belonging to the genus Tuber, include ectomycorrhizal species highly appreciated for their aroma. More than a hundred volatile organic compounds (VOCs) have been isolated from the fruiting bodies and mycelia of truffles (Zeppa et al., 2004; Splivallo et al., in press), and from ectomycorrhizal roots (Menotta et al., 2004). In this issue of New Phytologist, Splivallo et al. (pp. 417–424) demonstrate the first biological activity for truffle VOCs. These volatiles cause rapid and efficient leaf bleaching and root inhibition of Arabidopsis thaliana and are therefore regarded as phytotoxic compounds. These findings suggest that truffle volatiles may play a role in ‘burnt formation’, inhibition of herbaceous plants by truffles (Pacioni, 1991). ‘It can therefore be imagined that many more VOC-related inter- and intraorganismic interactions exist than previously thought.’ The importance of volatiles was underestimated in the past, but recently a series of interesting new findings have focused attention on these low-molecular-weight compounds with high vapour pressure. They are necessary for inter- and intraorganismic communication. Apparent VOC producers and receivers (e.g. flowers and pollinators, and male and female animals) have been investigated. However, it is necessary to reconsider the fact that, in principle, all living organisms have the potential to synthesize, emit and receive volatile compounds. It can therefore be imagined that many more VOC-related inter- and intraorganismic interactions exist than previously thought. Plants produce and emit diverse mixtures of VOCs which play key roles in above-ground and underground plant development and in plant–microbe and plant–insect interactions. Extensively investigated volatile signals of plants include methyl esters of the phytohormones jasmonic acid and salicylic acid, ethylene, terpenoids, and six-carbon volatiles (Pichersky et al., 2006; Tholl et al., 2006). In contrast, the influence of microbial volatiles on plant physiology is only beginning to be understood. Ryu et al. (2003, 2004) demonstrated that 2,3-butanediol and acetoin synthesized and emitted by plant growth-promoting Bacillus strains enhance growth and induce systemic resistance of A. thaliana. In truffles, Splivallo et al. observed that, depending on the Tuber species, fungal volatiles caused bleaching of A. thaliana leaves, and/or inhibition of root and leaf development. The finding that the truffle host plant Cistus incanus was similarly inhibited to A. thaliana indicates a rather widespread action profile for the volatiles. More importantly, these results raise the question of what possible role(s) these volatiles play in ecosystems, and whether they are indeed responsible for the formation of plant inhibitory zones (‘burnts’). That not only fungal, but also rhizobacterial volatiles can cause dramatic growth inhibitory effects on A. thaliana was recently demonstrated by Vespermann et al. (in press; Fig. 1). The volatile profiles of the rhizobacteria differ very much in qualitative and quantitative complexity; however, many compounds could not be identified, indicating that they comprise a new source of potential antibiotic compounds (Kai et al., 2007). In contrast, many truffle volatiles could be identified and individual compounds such as trans-2-octenal, 3-octenol and 1-octen-3-ol induced bleaching and root growth inhibition (Splivallo et al., in press). Interestingly when considering its biological activity, 1-octen-3-ol is responsible for the typical smell of fungi and thus is commonly found in the volatile blends of mushrooms. This compound together with trans-2-octenal led to a burst of hydrogen peroxide (H2O2) and to a significant increase of reactive oxygen species (ROS)-related enzyme activities in A. thaliana. Influence of rhizobacterial volatiles on the growth of Arabidopsis thaliana exposed to airborne chemicals released from growth-promoting Bacillus subtilis and growth-inhibiting Pseudomonas fluorescens strains. Figure courtesy of M. Kai, University of Rostock, Germany. Before the current work of Splivallo et al. with truffles, the phytohormone ethylene was the only characterized fungal VOC with implications in fungus–plant interactions. Botrytis cinerea is able to produce ethylene in vitro, and the emission of ethylene follows the pattern that is associated with hyphal growth (Cristescu et al., 2002). The possibility of 1-octen-3-ol–ethylene cross-talk would certainly be worth investigating in A. thaliana, as one of the functions of ethylene in plants is to adjust the release of H2O2 and other ROSs (Overmyer et al., 2000). The consequences of volatile production by bacteria for the target fungi are manifold; the volatiles affect the growth rate and the development of fungal mycelium and sclerotia, and lead to conidia deformation and discoloration (Wheatley, 2002; Kai et al., 2007). Most interest has been focused on the influence of fungal and bacterial volatiles on the mycelial growth of mycorrhizal fungi. Saprophytic fungi may cause growth depression in mycorrhizal fungi; for example, Trichoderma pseudokoningii volatiles inhibit Gigaspora rosea spore germination and mycorrhiza establishment in soybean (Glycine max) (Martinez et al., 2004). In contrast, spore germination and hyphal growth of Glomus mosseae were promoted by the volatiles of soil yeasts (Sampedro et al., 2004). This positive effect of yeast volatiles warrants further investigation. Bacterial volatiles, for example from Streptomyces spp., have been shown to promote the germination of G. mosseae spores (Tylka et al., 1991), and, depending on the fungal species, volatiles produced by mycorrhiza helper bacteria may either promote or inhibit mycelial growth (Garbaye & Duponnois, 1992). Direct contact among Paenibacillus sp. EJP73, Lactarius rufus and Scots pine (Pinus sylvestris) leads to accelerated mycorrhiza development. Using laboratory-based microcosms, Aspray et al. (2006) observed that volatile production by Paenibacillus sp. separated from plant roots led to inhibition of symbiosis development. These somewhat contradictory results reflect the general challenge of volatile application studies. Similarly, Barbieri et al. (2005) were able to analyse 65 volatiles produced by Staphylococcus pasteuri, an antagonist of Tuber borchii, while in S. pasteuri–T. borchii cocultures the spectrum of VOCs was distinct from that of the respective pure cultures. The terpenoid γ-pachoulene, a compound that was only produced in dual cultures, was linked to antifungal activity. Currently, no volatile profiles of the tri-trophic S. pasteuri–T. borchii–host plant interaction are known. From the increasing evidence accumulating in the literature, it has to be concluded that multifunctional and multiorganismic volatile-based interactions exist in ecosystems, probably to a greater extent than has been envisioned. In the light of the observations of Splivallo, Vespermann, Ryu and colleagues showing that truffle or rhizobacterial VOCs directly influence plants (including host plants), one possible next step would be to expand investigations to other potential partners in the interaction; for example, to test how the truffle volatiles influence mycorrhiza development (Menotta et al., 2004) or how rhizobacterial volatiles influence the soil ecosystem by studying volatile-dependent interactions between phytopathogenic fungi, plants, nematodes, etc. In this context, the volatiles of mycorrhiza helper bacteria and mycorrhiza-associated fungi can now be seen from a new perspective regarding symbiosis establishment. A more specific question relates to the ecological relevance of the truffle volatile–host plant interaction: are the bioactive volatiles responsible for the inhibition of herbaceous plants in nature? Such questions can now be addressed, for example, by relating VOC dose–plant responses to measurements within microcosms. The rapid development of analytical methods and the ever-increasing number of accessions in molecular mass databases, together with the use of model organisms, suggest that we have a realistic chance of taking a leap forward in our understanding of the role of volatiles in microbe–plant interactions in the near future.