More complexity in the mycorrhizal world

Authors

  • M.-A. Selosse,

    Corresponding author
    1. CEFE-CNRS, UMR 5175, Equipe Interactions Biotiques, 1919 Route de Mende, F-34 293 Montpellier cedex 5, France;
      (*Author for correspondence: tel +33(0)4 67 61 32 31; fax +33(0)4 67 41 21 38; email ma.selosse@wanadoo.fr)
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  • S. Duplessis

    1. UMR 1136 INRA/UHP-Nancy 1, Interactions Arbres Micro-organismes, Centre INRA de Nancy, F-54280 Champenoux, France
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(*Author for correspondence: tel +33(0)4 67 61 32 31; fax +33(0)4 67 41 21 38; email ma.selosse@wanadoo.fr)

5th International Conference on Mycorrhiza, Granada, Spain, July, 2006

The 5th International Conference on Mycorrhiza (ICOM5) was held in Granada (Andalucia, Spain), a multicultural city with a heritage that is both Arabic and European. Over 650 mycorrhizal scientists from more than 50 countries attended the meeting and took part in the varied social events, such as the night visit to the Alhambra (Fig. 1) and the traditional ‘Wines of the World’ contest. All of which contributed towards making this an open and friendly meeting, enhancing exchange and discussion of the current progress in this fast-moving field. This ICOM edition was thus very successful.

Figure 1.

ICOM5 participants visited the Alhambra at night. This series of palaces and gardens, built under the Nazari dynasty in the 14th century, introduced mycorrhizologists to complexity (here, wall plasterwork ornaments), a complexity reminiscent of that of mycorrhizal networks, fungal speciation and genes networks. (Photograph by M.-A. Selosse.)

‘The Laccaria genome is thus strikingly multifaceted, with Martin comparing it to the two-headed roman god Janus’

As the conference opened, the Joint Genome Institute (JGI, Walnut Creek, CA, USA; US Department of Energy, USA) launched the genome sequence portal (http://genome.jgi-psf.org/Lacbi1/Lacbi1.home.html) for the mycorrhizal fungus, Laccaria bicolor. This being the official release of the first complete genome for a mycorrhizal fungus made it one of the major breakthroughs widely discussed during this meeting. Ecology and evolution dominated among the posters as well as in the sessions and workshops and, as usual, most talks focused on arbuscular mycorrhizas (AMs, involving Glomeromycetes that form arbuscular haustoria in host cells) and ectomycorrhizas (ECMs, associating Ascomycetes and Basidiomycetes with trees and shrubs). ECMs only half as popular as AMs among talks on cellular, molecular and genomic aspects of the symbiosis, but both were equally represented among talks on ecology and evolution. Such an overrepresentation of ECMs, as compared with their relatively smaller host spectrum and geographic breadth, probably arises because they are easily tractable and because most research groups work in temperate regions, where they dominate. It was noteworthy, with a few exceptions, that tropical mycorrhizal models were also underrepresented; however, given that ICOM6 will be held in Brazil in 2009 it is hopeful that tropical models will have a much larger prominence there!

The dawn of genomics in the mycorrhizal world

Availability of genome sequences from ecologically and taxonomically diverse fungi will allow ongoing research into these species and enhance the value of other sequences through comparative studies of gene and genome evolution, metabolic and regulatory pathways, as well as symbiosis and pathogenesis. Francis Martin (INRA Nancy, France) summarized the major features of the 65 megabase L. bicolor genome obtained by a whole-shotgun sequencing strategy in 2005. The International Ectomycorrhizal Genome Consortium (http://mycor.nancy.inra.fr/IMGC/) had brought together experts from various research areas to define the contours of the most important gene families. From > 20 000 putative genes identified so far, Martin pinpointed: (i) the important number of signalling pathway genes; (ii) the numerous genes coding short polypeptides (about 3000 genes encoding proteins of < 100 amino acids, including several secreted proteins); and (iii) the so-called (by Martin) ‘saprotrophic’ machinery, as Laccaria possesses most functions necessary for a saprotrophic lifestyle, as compared with Coprinus cinereus or Phanerochaete crysosporium. This last point is particularly interesting with regard to the hypothesis that ECM fungal clades experienced reversals to the saprotrophic lifestyle during their evolution (Hibbet et al., 2000). Besides, several genes falling into the signalling and secretome categories have homologues involved in pathogenesis in plant pathogenic basidiomycetes. The Laccaria genome is thus strikingly multifaceted, with Martin comparing it to the two-headed roman god Janus. A major challenge will be to determine the role of such genes in symbiosis, using genetic transformation techniques now available for L. bicolor (Kemppainen et al., 2005).

Peter Lammers (New Mexico State University, NM, USA) presented the sequencing project of the AM Glomus intraradices, sequenced by the JGI along with Laccaria in a project focusing on microbes interacting with Populus trichocarpa (Martin et al., 2004; Tuskan et al., 2006). Because of its obligate biotrophy, obtaining sufficient amounts of uncontaminated high-molecular-weight DNA had been a major problem. Low content in GC nucleotides (c. 30%) also biased routine cloning and delayed sequencing but the JGI was finally able to obtain a draft of the genome sequence (4 × coverage). Analysis of this second genome will help towards understanding what allows a fungus to build a long-lasting, beneficial interaction with plants. Surprisingly, Lammers reported the expression of a homologue of Dmc1 (disruption of meiotic control1), a gene involved in meiotic recombination, in G. intraradices germinating spores. The finding of homologues for two more meiotic genes, Msh4 and Msh5, and mating type genes by Hélène Niculita and Alexandre Colard from Ian Sanders’ group (University of Lausanne, Switzerland) raised the question of the role of such genes in fungi considered as asexual.

ECM nutrition and transport in a genomic perspective

Uwe Nehls (University of Tübingen, Germany) addressed the role of the plant nutritional status on development of the ECM symbiosis. He has identified families of sugar transporters in poplar and ECM fungi, and described the regulation of their expression during ECM formation regarding different nutrient supplies. His results supported an interesting hypothesis that emphasizes the permissive role of carbohydrate flux in mycorrhizal development, and this can be brought together with the hypothesis for a link between nutrient fluxes and the development of the AM symbiosis (Fitter, 2006). Michel Chalot (University of Nancy, France) presented the annotation and characterization of nitrogen mobilization and transport enzymes families of L. bicolor and P. trichocarpa, reporting on the expansion of some gene families in Laccaria as compared with other fungi. Both talks highlighted the strength of genomics that allows gene fishing in genome sequences (Martin et al., 2004; Tuskan et al., 2006). Access to the genomes of both AM and ECM fungi and a common partner tree will open up greater possibilities for the study of mycorrhizal biology in future years and the next ICOM should be particularly exciting in this regard. A synergy can be expected with the parallel development of new techniques announced by several groups, such as the use of laser microdissection techniques to get closer to the molecular events in the different plant or fungal compartments, or the refinement of microscopy fluorescent techniques (Lohse et al., 2006).

Early signalling events in the AM symbiosis

Another breakthrough since the previous ICOM was the end of the ‘Branching Factor’ (BF) race. Kokhi Akiyama (Osaka University, Japan) and Nathalie Séjalon-Delmas (CNRS, Toulouse University, France) presented independent works that led to identification of such molecules in AM plants (Akiyama et al., 2005; Besserer et al., 2006). BF consists of strigolactones that are recognized by AM fungi at the presymbiotic stage and trigger hyphal branching. These groups now work on the identification of the Myc factor (MF), the fungal signal molecule triggering plant responses for a successful fungal colonization, and Akiyama expressed his hope to identify MF by the next ICOM. Séjalon-Delmas showed the effect of sorgolactone (Sorghum BF) and two chemical analogues from the AM Gigaspora rosea (Besserer et al., 2006). A transcriptome analysis conducted on fungal spores subjected to BF reveals specific effects on mitochondrial biogenesis and lipid catabolism after signal perception at the presymbiotic stage. Other presentations on different sides of the AM symbiosis showed promising progress in the molecular dissection of the early events of symbiosis development, although large gaps still remain.

Cryptic species and fungal ecology and evolution

Classical species concepts in ECM fungi are mostly based on fruitbody (or vegetative) morphology. Molecular tools now allow delineation of biological species, that is groups of individuals among which molecular markers are shared and recombine. The fingerprint of recombination is that phylogenies of individuals within the same biological species are not congruent for different loci. In contrast to this, phylogenetic trees of different loci are expected to be congruent between species. The point at which congruence between phylogenetic trees of different loci arises therefore delineates biological species (Douhan & Rizzo, 2005). This was successfully used to show that the widespread ECM Cenococcum geophilum encompasses cryptic species, that is biological species undistinguished by ECM or sclerotia morphology (Douhan & Rizzo, 2005). Additional molecular dissections of ECM fungal species were reported at ICOM5, for example in ‘Tricholoma scalpturatum’ (Gryta et al., 2006) and among Xerocomus spp., for which Andy Taylor (Department of Forest Mycology, Uppsala, Sweden) showed an underestimated cryptic species diversity, together with an overestimated species overlap between continents. However, mechanisms for evolutionary rise of cryptic species remain unclear.

Studies on mycorrhizal fungal diversity now face a problem familiar to marine microbiologists, the so-called Hutchinson's ‘paradox of the plankton’: why are there so many species, apparently sharing the same niche, when competitive exclusion predicts extinction of the least competitive ones? Should we imagine that so many different niches exist, for example because of host diversity? Indeed, Xerocomus cryptic species vary in specificity (Taylor et al., 2006), making host specialization a possible motor for their emergence – but evidence for such specificity is lacking in other cases, and host-poor ecosystems still harbour considerable fungal diversity. Soil heterogeneities might favour large niche diversity. For niche delimitation, as well as a better understanding of the functional implication of ECM species diversity, enzymatic profiling is also a promising tool (Courty et al., 2005). In this approach, microplate tests measure several enzymatic activities on excised ECM root tips, offering clever insights into fungal resource utilization over time and space. Alternatively, fluctuations or unpredictability in ecosystems might allow coexistence of species with redundant niches, just by limiting population below any competition. Peter Kennedy (University of California, Berkeley, CA, USA) reported new laboratory and field-based evidence of competitive priority effects as well as hierarchies among a suite of closely related ECM Rhizopogon species (Kennedy & Bruns, 2005), so biotic interactions also contribute towards shaping different communities. Data on competition between mycorrhizal fungi are still rare, and the ‘paradox of mycorrhizal diversity’ remains intriguing.

Also noteworthy is that spatial scaling of mycorrhizal community richness might differ from that of free-living microbes. Investigating islands of ECM trees separated by non-ECM plant communities, Kabir Peay (University of California, Berkley) reported that the slope of the relationship between species number and island area is more similar to plants and other macro-organisms than the slopes usually associated with microbes (approx. three to six times lower). Symbiosis might shape ECM communities, and thus their evolution. Indeed, James Bever (Indiana University, Bloomington, IN, USA) provided the first convincing evidence that plants can choose their AM partners under some conditions: in a split root system separately inoculated with two AM strains (one mutualistic, the other less beneficial), the mutualistic fungus received more carbon and had increased fitness, as measured by spore production. Interestingly, however, the better strain was the worse competitor, so the spatial dynamics of root colonization may also be important in determining mycorrhizal species richness at small spatial scales. Besides stabilization of the mutualism in mycorrhizal interaction (Kiers & van der Heijden, 2006), such plant sanctions on less rewarding mycorrhizal partners might strongly contribute to fungal evolution.

Mycorrhizal networks: linking plants and shaping communities?

An individual fungal mycelium can associate with two or more plants, which are not conspecific, resulting in a common mycorrhizal network (CMN) linking plants (Selosse et al., 2006). Several descriptive works currently focus on extraradical mycelium, the ‘linking part’ of CMNs. Ian Anderson (The Macaulay Institute, Aberdeen, Scotland) reported on the fine-scale distribution and repartition of extraradical mycelia and ECM root tips by ITS analysis. This work revealed some discrepancies (Genney et al., 2006); for example, extraradical mycelia can be found in soil layers different from those containing ECM root tips and seven times as many species were found among extraradical mycelia than on root tips. At the functional level, Jonathan Leake (University of Sheffield, UK) reported that, in a grassland ecosystem, AM fungi represent one-third of soil microbiota and respire 5–9% of the carbon fixed by host plants within 34 h.

Leanne Philip (University of British Columbia, Vancouver, BC, Canada) re-investigated carbon transfers through CMNs previously reported by Simard et al. (1997). Where Pseudotsuga menziesii (Douglas-fir) and Betula papyrifera (paper birch) seedlings were grown together in pot systems or root chambers, transfer intensity was greater, (i) in the presence of shared ECM fungi (vs noninoculated cultures), and (ii) when hyphal connections were intact (vs severed). In the field, CMNs comprising three ECM species (paper birch, Douglas-fir, Larix occidentalis) showed reversion of the net flux over the year (to B. papyrifera in spring and autumn, when P. menziesii photosynthesis was higher, and to Douglas-fir in summer when paper birch photosynthesis was higher), suggesting that such transfers could be more cooperative than previously thought. Stable isotopes are also major tools for demonstrating carbon transfers: since 13C content of ECM fungi differs from that of autotrophic plants, the contribution of fungal carbon to plant biomass can be quantified without artificial labelling. Gerhard Gebauer (University of Bayreuth, Germany) reported that several orchid species (e.g. Julou et al., 2005) and other understorey green plants from ECM temperate forests partly rely on fungal carbon in addition to photosynthesis, a strategy called ‘mixotrophy’ (Selosse et al., 2006). Since their mycorrhizal fungi form ECM on surrounding trees, a CMN is likely to transfer nutrients from overstorey trees to the mixotrophs. However, carbon transfers were received with more scepticism by AM ecologists. Although, as Jonathan Leake recalled, some purely heterotrophic AM plants exclusively use this carbon source, AM mixotrophs are hitherto unknown. Carbon transferred from fungi to the host plant could arise from another source; Jean Garbaye (INRA Nancy, France) suggested that carbon heterotrophically obtained by Lactarius quietus could contribute to the unbalanced spring carbon budget in Quercus spp. Indeed, L. quietus ECM tips undergo a flush of enzymes degrading complex substrates, at a time where carbon budget of the tree seems in deficit. As a dramatic example, an achlorophyllous Japanese orchid was reported to be mycorrhizal with saprophytic Coprinaceae (Yamato et al., 2005). So, how often is dead soil carbon available to plants through mycorrhizal fungi?

Common mycorrhizal networks also act by providing an inoculum to seedlings. Michael Booth (University of Alaska at Yale, CT, USA) reported that ECMs limit the effect of competition of overstorey trees on seedlings (Booth, 2004). Although nutrient transfer is unlikely to be involved (as shown by analysis of 13C content), CMNs provide a diverse ECM inoculum. Kazuhide Nara (University of Tokyo, Japan) showed experimental and circumstantial evidence that, in primary successions on volcanic soils, ECM fungi recruited by Salix reinii facilitate settlement of conspecifics (Nara, 2006) and other species associated with ECM fungi, such as Betula, Larix or Pyrola species. CMNs therefore drive ecological successions, among other soil factors (Kardol et al., 2006).

Finally, mycorrhizas integrate into even larger networks of interactions: Wim van der Putten (Netherlands Institute of Ecology, Heteren, the Netherlands) showed the importance of looking at multitrophic interactions, including above- and below-ground biota linked by plants. For example, plant defence above ground is strongly influenced by below-ground interactions (Bezemer et al., 2005), among which mycorrhizas play a significant role. Stefan Reidinger (University of London, UK) showed how mycorrhizas can lead to changes in the structure of herbivore insect communities (Gange et al., 1999). Most multitrophic interactions reported at ICOM5 focused on mycorrhizospheric bacteria, making frequent use of analysis of phospholipid fatty acids to describe the fungal and bacterial diversity at high systematic level.

Enlarging the spectrum of interacting organisms and using the genomes of model symbioses will probably enhance the complexity, but also the physiological and ecological relevance, of mycorrhizal research. More species from different fungal clades are currently on the way to being sequenced. Perhaps in the future, increased overlap of once disparate fields within mycorrhizal research will be possible, for example the fusion of ecological and genomic research. Bringing field and in silico investigations together in common ‘ecogenomics’ sessions may be what we should expect at ICOM6!

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