Francis Martin

Mycorrhizal ecology and evolution: the past, the present, and the future

Almost all land plants form symbiotic associations with mycorrhizal fungi. These below-ground fungi play a key role in terrestrial ecosystems as they regulate nutrient and carbon cycles, and influence soil structure and ecosystem multifunctionality. Up to 80% of plant N and P is provided by mycorrhizal fungi and many plant species depend on these symbionts for growth and survival. Estimates suggest that there are c. 50 000 fungal species that form mycorrhizal associations with c. 250 000 plant species. The development of high-throughput molecular tools has helped us to better understand the biology, evolution, and biodiversity of mycorrhizal associations. Nuclear genome assemblies and gene annotations of 33 mycorrhizal fungal species are now available providing fascinating opportunities to deepen our understanding of the mycorrhizal lifestyle, the metabolic capabilities of these plant symbionts, the molecular dialogue between symbionts, and evolutionary adaptations across a range of mycorrhizal associations. Large-scale molecular surveys have provided novel insights into the diversity, spatial and temporal dynamics of mycorrhizal fungal communities. At the ecological level, network theory makes it possible to analyze interactions between plant-fungal partners as complex underground multi-species networks. Our analysis suggests that nestedness, modularity and specificity of mycorrhizal networks vary and depend on mycorrhizal type. Mechanistic models explaining partner choice, resource exchange, and coevolution in mycorrhizal associations have been developed and are being tested. This review ends with major frontiers for further research.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis

Significance The arbuscular mycorrhizal symbiosis between fungi of the Glomeromycota phylum and plants involves more than two-thirds of all known plant species, including important crop species. This mutualistic symbiosis, involving one of the oldest fungal lineages, is arguably the most ecologically and agriculturally important symbiosis in terrestrial ecosystems. The Glomeromycota are unique in that their spores and coenocytic hyphae contain hundreds of nuclei in a common cytoplasm, which raises important questions about the natural selection, population genetics, and gene expression of these highly unusual organisms. Study of the genome of Rhizophagus irregularis provides insight into genes involved in obligate biotrophy and mycorrhizal symbioses and the evolution of an ancient asexual organism, and thus is of fundamental importance to the field of genome evolution. The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

The rhizosphere zoo: An overview of plant-associated communities of microorganisms, including phages, bacteria, archaea, and fungi, and of some of their structuring factors

Rhizosphere microorganisms have two faces, like Janus the Roman god of gates and doors who symbolizes changes and transitions, from one condition to another. One face looks at the plant root, the other sees the soil. The ears and the nose sense the other gods around and the mouths are wide open, swallowing as much as they can, and as described in Chapter 11, they also are busy talking. These faces may as well represent Hygieia (the Greek god of Health and Hygiene, the prevention of sickness and the continuation of good health) and Morta (the Roman god of death) for rhizosphere microbes can be beneficial, and promote plant growth and well being (Chapter 12) or detrimental, causing plant sickness and death (Chapter 13). It can be argued that many rhizosphere microbes are “neutral”, faceless saprophytes that decompose organic materials, perform mineralization and turnover processes. While most may not directly interact with the plant, their effects on soil biotic and abiotic parameters certainly have an impact on plant growth. Maybe they are Janus’ feet, the unsung heroes of the rhizosphere. This chapter addresses some aspects of the taxonomical and functional microbial diversity of the rhizosphere. Bacteria, Archea, viruses and Fungi will be at the heart of our discussion, while other rootassociated eukaryotes are the subjects of other chapters

Soil networks become more connected and take up more carbon as nature restoration progresses

Soil organisms have an important role in aboveground community dynamics and ecosystem functioning in terrestrial ecosystems. However, most studies have considered soil biota as a black box or focussed on specific groups, whereas little is known about entire soil networks. Here we show that during the course of nature restoration on abandoned arable land a compositional shift in soil biota, preceded by tightening of the belowground networks, corresponds with enhanced efficiency of carbon uptake. In mid- and long-term abandoned field soil, carbon uptake by fungi increases without an increase in fungal biomass or shift in bacterial-to-fungal ratio. The implication of our findings is that during nature restoration the efficiency of nutrient cycling and carbon uptake can increase by a shift in fungal composition and/or fungal activity. Therefore, we propose that relationships between soil food web structure and carbon cycling in soils need to be reconsidered.

High genetic diversity in a population of the ectomycorrhizal basidiomycete laccaria amethystina in a 150-year-old beech forest

The genetic structure of a population of the ectomycorrhizal basidiomycete Laccaria amethystina (Bolt. ex Hooker) Murr. was assessed in a closed 150‐year‐old beech (Fagus sylvatica L.) forest in the Vosges Mountains in northeastern France. During the autumn of 1994 and 1997, sporophores were collected from three 100‐m2 sampling plots located along a 120‐m transect crossing the beech stand. The genetic variation of 676 sporophores was initially estimated using heteroduplex analysis of the ribosomal DNA intergenic spacer (IGS1). Ten unique IGS1 heteroduplex/homoduplex patterns were identified, although three types represented most of the sporophores analysed. Each group of IGS1 type was then analysed using random amplified microsatellite analysis (RAMS). RAMS resolved 388 different genotypes amongst the 634 sporophores analysed from the three plots during the autumn of 1994 and 1997. Density as high as 130 genets per 100 m2 was observed during the autumn of 1994. The largest clone covered ≈ 1 m2, but most genets covered a few cm2 and produced only one to three sporophores. Only eight genotypes identified in 1994 were found in 1997. Although L. amethystina has the capacity for vegetative persistence, the present study indicates that its populations maintain a genetic structure more consistent with a high frequency of sexual reproduction. This suggests that beech trees could be recolonized by new genotypes each year. Alternatively, this spatial distribution may also arise from erratic fruiting of underground persistent genets. These features (i.e. numerous genets of small size), typical of ruderal species, contrast with studies carried out on other ectomycorrhizal basidiomycetes occurring in mature closed forests.

Functional Assays and Metagenomic Analyses Reveals Differences between the Microbial Communities Inhabiting the Soil Horizons of a Norway Spruce Plantation

In temperate ecosystems, acidic forest soils are among the most nutrient-poor terrestrial environments. In this context, the long-term differentiation of the forest soils into horizons may impact the assembly and the functions of the soil microbial communities. To gain a more comprehensive understanding of the ecology and functional potentials of these microbial communities, a suite of analyses including comparative metagenomics was applied on independent soil samples from a spruce plantation (Breuil-Chenue, France). The objectives were to assess whether the decreasing nutrient bioavailability and pH variations that naturally occurs between the organic and mineral horizons affects the soil microbial functional biodiversity. The 14 Gbp of pyrosequencing and Illumina sequences generated in this study revealed complex microbial communities dominated by bacteria. Detailed analyses showed that the organic soil horizon was significantly enriched in sequences related to Bacteria, Chordata, Arthropoda and Ascomycota. On the contrary the mineral horizon was significantly enriched in sequences related to Archaea. Our analyses also highlighted that the microbial communities inhabiting the two soil horizons differed significantly in their functional potentials according to functional assays and MG-RAST analyses, suggesting a functional specialisation of these microbial communities. Consistent with this specialisation, our shotgun metagenomic approach revealed a significant increase in the relative abundance of sequences related glycoside hydrolases in the organic horizon compared to the mineral horizon that was significantly enriched in glycoside transferases. This functional stratification according to the soil horizon was also confirmed by a significant correlation between the functional assays performed in this study and the functional metagenomic analyses. Together, our results suggest that the soil stratification and particularly the soil resource availability impact the functional diversity and to a lesser extent the taxonomic diversity of the bacterial communities.

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors

Summary Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose‐degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition ‘toolbox’ has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Temporal persistence and spatial distribution of an American inoculant strain of the ectomycorrhizal basidiomycete Laccaria bicolor in a French forest plantation

Selected strains of ectomycorrhizal fungi, such as the basidiomycete Laccaria bicolor, are currently being used as inoculants in nurseries to improve growth of forest trees after outplanting. Information is needed on the survival of these introduced strains in forests and their impact on indigenous biodiversity. Dissemination and persistence of an American strain, L. bicolor S238N, were studied 10 years after outplanting in a Douglas fir plantation located at Saint‐Brisson (Morvan, France). About 430 Laccaria spp. sporophores were collected over 3 years. Inheritance of nuclear ribosomal DNA, as well as RAPD markers, was characterized in L. bicolor S238N, using a haploid progeny set of 91 monokaryons. More than 50 markers were identified (19 heterozygous and 33 homozygous or cytoplasmic markers), which unambiguously confirmed that the introduced strain was still present in the inoculated plots. Neither selfing (P < 0.0008) nor introgression with indigenous strains was detected although in vitro interfertility between the American strain and indigenous L. bicolor was identified. No ingress of the introduced genet into adjacent uninoculated plots colonized by various local Laccaria genets was detected. It is proposed that the spatial distributions identified have developed through mycelial propagation of the introduced strain and intraspecific competition with native genets. Although longer‐term data is still lacking, the stability of the inoculant strain and the limited disturbance to indigenous populations described support large‐scale nursery production of this host‐fungal combination.

Monitoring the persistence of Laccaria bicolor as an ectomycorrhizal symbiont of nursery-grown Douglas fir by PCR of the rDNA intergenic spacer.

The large‐scale inoculation of selected beneficial ectomycorrhizal fungi in forest nurseries has generated renewed interest in the ecology of these symbiotic fungi. However, information on the dissemination and persistence of introduced symbionts is scarce due to the limitation of the current identification methods. To identify ectomycorrhizal fungi on single root tips, we investigated the polymorphism of the PCR‐amplified ribosomal DNA intergenic spacer (IGS) from a wide range of ectomycorrhizal fungi. To investigate the reliability of this molecular approach in large‐scale surveys, the dissemination and persistence on Douglas fir seedlings of the introduced Laccaria bicolor S238N were assessed in a forest nursery in the Massif Central (France). Several hundred ectomycorrhizas and fruiting bodies were sampled from plots where control and L. bicolor inoculated‐Douglas fir seedlings were grown for 1.5 years. PCR typing of mycorrhizas indicated that trees inoculated with L. bicolor S238N remained exclusively colonized by that isolate (or sexually derived isolates) for the entire test period. In contrast, control seedlings were infected by indigenous isolates of Laccaria laccata and Thelephora terrestris. The molecular evidence for the persistence of the introduced mycobiont despite the competition from indigenous isolates of the same species provides further illustration of the potential of exotic species for large‐scale microbial application.