Roland Marmeisse

Fine‐scale structure of populations of the ectomycorrhizal fungus Hebeloma cylindrosporum in coastal sand dune forest ecosystems

The basidiomycete mushroom Hebeloma cylindrosporum is a frequently found pioneer ectomycorrhizal species naturally associated with Pinus pinaster trees growing in coastal sand dune ecosystems along the Atlantic south‐west coast of France. The genotypic diversity and spatial structure of three populations of this fungal species have been studied. At each site the basidiocarps were mapped, sampled and propagated as pure mycelial cultures. For each of the isolates, we have studied polymorphisms in the mitochondrial genome, polymorphisms at two different nuclear loci and also fingerprints produced with a multicopy DNA probe. The comparison of the different polymorphisms obtained, with each of the four molecular methods used, allowed the identification of several of the different genets present in each site. In two of the studied sites most of the basidiocarps, which often occurred as dense patches of 10–30 in 1 m2 or less, were of a unique genotype, suggesting the below‐ground mycelia to be of a small size (from 50 cm2 to approx. 7 m2 for the larger mycelia) and that the root system of a single Pinus tree can host several genets of the same symbiotic fungus. In the two sites, which were studied again after a 3‐year interval, none of the genotypes identified in the first year of sampling was re‐identified 3 years later. These results contrast with those reported for other species of soilborne homobasidiomycete species, either ectomycorrhizal, parasitic or saprophytic, showing mostly large clones resulting from the vegetative growth and from persistence of below‐ground mycelia. Sexual reproduction through meiospore dispersal seems to play a key role in the structuring of the populations of H. cylindrosporum. Mycelia associated with the root systems seem to be replaced after 1 or a few years, during which basidiocarp differentiation takes place. As opposed to the few other studied ectomycorrhizal species, H. cylindrosporum has the characteristics of ruderal species, with a short life‐span adapted to pioneer situations, e.g. to nutrient‐poor and unstable sandy soils of coastal sand dunes.

Characterisation and expression analysis of a nitrate transporter and nitrite reductase genes, two members of a gene cluster for nitrate assimilation from the symbiotic basidiomycete Hebeloma cylindrosporum

Abstract Symbiotic ectomycorrhizal fungi contribute to the nitrogen nutrition of their host-plants but little information is available on the molecular control of their nitrogen metabolism. We cloned and characterised genes encoding a nitrite reductase and a nitrate transporter in the ectomycorrhizal basidiomycete Hebeloma cylindrosporum. These two genes are divergently transcribed and linked to a previously cloned nitrate reductase gene, thus demonstrating that nitrate assimilation gene clusters occur in homobasidiomycetes. The nitrate transporter polypeptide (NRT2) is characterised byxa012 transmembrane domains and presents both a long putative intracellular loop and a short C-terminal tail, two structural features which distinguish fungal high-affinity transporters from their plant homologues. In different wild-type genetic backgrounds, transcription of the two genes was repressed by ammonium and was strongly stimulated not only in the presence of nitrate but also in the presence of organic nitrogen sources or under nitrogen deficiency.

Correspondence between genet diversity and spatial distribution of above- and below-ground populations of the ectomycorrhizal fungus Hebeloma cylindrosporum

Population studies of ectomycorrhizal fungal species have largely relied upon fruit body (the reproductive organ) sampling. Analysis of the fruit bodies alone supposes that they reflect the present and spatial organization of all below‐ground genets (mycorrhizas and extramatrical mycelia). The relation between fruit bodies and ectomycorrhizas was investigated for the basidiomycete agaric Hebeloma cylindrosporum in four Pinus pinaster stands in south‐west France. Genet identification was based on the comparison of polymorphisms within a hypervariable segment of the ribosomal intergenic spacer amplified by polymerase chain reaction (PCR) using a H. cylindrosporum species‐specific primer. Mycorrhizas were sorted from soil samples collected underneath patches of fruit bodies or patches where fruit bodies had or had not been observed during the years prior to mycorrhiza collection. On average 65% of the 1026 mycorrhizas collected underneath fruit bodies were formed by H. cylindrosporum, whereas only 2% of the 954 collected in places from where fruit bodies were absent were formed by this species. All genotypes identified above ground were also identified below ground. In patches where one genotype formed all or more than 90% of the fruit bodies, the same genotype formed all or a large majority of the mycorrhizas. In patches occupied by several different fruiting genotypes, additional nonfruiting ones could be present on the root systems. In all cases, the mycorrhizas of one genotype were found no more than 10–20 cm away from its corresponding fruit bodies, and fruit body disappearance at a given place was associated with the disappearance of the corresponding mycorrhizas within 1 year. Although there was not a strict coincidence between the total numbers of genets present below ground and of those forming fruit bodies, fruit body analysis for H. cylindrosporum appears to reflect both the genetic diversity and the spatial structure of its below‐ground populations. The results obtained also illustrate the rapid turnover of ectomycorrhizal fungal species on the root systems in the absence of any obvious major disturbance of the ecosystem.

Spatial distribution of the below-ground mycelia of an ectomycorrhizal fungus inferred from specific quantification of its DNA in soil samples.

In natural forest ecosystems several ectomycorrhizal fungal species cohabit on host plant root systems. To evaluate the ecological and functional impact of each species, it is necessary to appreciate the distribution and abundance of its mycelia in the soil. We developed a competitive PCR (cPCR) method for the basidiomycete Hebeloma cylindrosporum that allows quantification of its DNA in complex DNA mixtures extracted directly from soil samples. The target sequence chosen for the cPCR analysis was a 533-bp fragment of the nuclear ribosomal intergenic spacer, amplified using two species-specific primers. The detection threshold of the cPCR protocol developed was 0.03 pg of genomic DNA. This method was applied to soil samples collected from beneath and at various distances from a group of fruit bodies in a Pinus pinaster forest stand. The results revealed that H. cylindrosporum below-ground biomass was concentrated directly underneath the fruit bodies or very close to them, while no DNA of this species could be detected in soil samples collected at more than 50 cm away. In the vicinity of fruit bodies, H. cylindrosporum soil DNA concentration varied considerably (between 10 and 0.07 ng g soil(-1)) and decreased sharply with increased distance from the fruit bodies. This work demonstrates the potential of competitive quantitative PCR for the study of the distribution, abundance and persistence of the mycelia of an ectomycorrhizal fungal species in soil.

Population dynamics of the symbiotic mushroom Hebeloma cylindrosporum: mycelial persistence and inbreeding

The pattern of colonization of a forest site by the ectomycorrhizal basidiomycete fungus Hebeloma cylindrosporum Romagnesi was followed from 1993 until 1997. Fruit-bodies of this tetrapolar heterothallic species were mapped, collected and propagated as pure mycelial cultures. Isolates were analysed for their mating-types and molecular markers (rDNA polymorphism and RAPD). Dedikaryotization of the 26 isolates collected in 1993 and the separate analysis of each individual haploid nucleus established that two fully compatible genets, which occupied two nonoverlapping territories, were present. Isolates belonging to the same genet could nevertheless be distinguished from each other based on Southern hybridization using hyperpolymorphic DNA probes. A majority of the 143 isolates collected from 1994 to 1997 belonged to either of the two genets identified in 1993, whose territories extended at a rate of about 0.45–0.60 m per year. Selfing of the two 1993 genets, rather than outcrossing, was the most likely explanation for the origin of additional genotypes identified between 1995 and 1997. The spatial distribution of fruit-bodies and genets of H. cylindrosporum suggested that only a fraction of the sampled area was favourable to colonization and that genetic diversification through meiospore dispersal may be inhibited by the presence of resident genets, possibly via a somatic incompatibility system.

Intraspecific variation in use of different organic nitrogen sources by the ectomycorrhizal fungus Hebeloma cylindrosporum

The ectomycorrhizal (ECM) fungus Hebeloma cylindrosporum is an appropriate model to study the intraspecific functional diversity of ECM fungi in forest ecosystems. Numerous metabolic genes, specifically genes related to nitrogen assimilation, have been characterised for this species and the spatial and temporal structures of its natural populations have been extensively worked out. In this paper, we reveal the extent to which intraspecific variation exists within this fungus for the ability to use organic nitrogen, an important functional characteristic of ECM fungi. In addition to ammonium and nitrate, H. cylindrosporum can use at least 13 different amino acids out of 21 tested as sole nitrogen source, as well as urea and proteins. By screening 22 genetically different wild type haploid strains we identified obvious differences in use of six nitrogen sources: alanine, glycine, phenylalanine, serine, bovine serum albumin and gelatine. Of the 22 haploid strains, 11 could not use at least one of these six nitrogen sources. The inability of some haploid strains to use a nitrogen source was found to be a recessive character. Nevertheless, obvious differences in use of the four amino acids tested were also measured between wild type dikaryons colonising a common Pinus pinaster root system. This study constitutes the basis for future experiments that will address the consequences of the functional diversity of an ECM fungus on the functioning of the ECM symbiosis under natural conditions.

Fungal ectomycorrhizal community and drought affect root hydraulic properties and soil adherence to roots ofPinus pinaster seedlings

Pinus pinaster seedlings were grown in a sandy dune soil either inoculated withHebeloma cylindrosporum or let to natural colonisation. Six months later, half of the seedlings of both treatments were subjected to a 3-week moderate drought. Root colonisation analysis showed that root tips were colonised to almost 100% independent of the inoculation. DNA determination of the ectomycorrhizal morphotypes showed that inoculated seedlings were extensively mycorrhized byH. cylindrosporum (more than 75%) whereas non-inoculated seedlings were mycorrhized by the exotic speciesThelephora terrestris (50%) andLaccaria bicolor (30%) and to a lesser extent byH. cylindrosporum (20%). Drought did not affect these frequencies. Total plant biomass was not affected by the mycorrhizal status or by drought but the root/shoot biomass ratio as well as the root/leaf surface area ratio were much lower in seedlings extensively colonised byH. cylindrosporum. Root hydraulic conductivity was higher in plants mainly mycorrhized byH. cylindrosporum, showing that this fungus improved the water uptake capacity of the root system as compared toT. terrestris and/orL. bicolor. This positive effect was also found under drought but to a lesser extent.H. cylindrosporum also increased the amount of root-adhering soil as compared to the other fungal symbionts, illustrating the performance of this association in aggregating sandy soil particles and developing the rhizosheath. The origin of the reduced root hydraulic resistance byH. cylindrosporum mycorrhization is discussed for the whole path including soil, soil-root interface and root cortex.

Discovering Protein-Coding Genes from the Environment: Time for the Eukaryotes?

Eukaryotic microorganisms from diverse environments encompass a large number of taxa, many of them still unknown to science. One strategy to mine these organisms for genes of biotechnological relevance is to use a pool of eukaryotic mRNA directly extracted from environmental samples. Recent reports demonstrate that the resulting metatranscriptomic cDNA libraries can be screened by expression in yeast for a wide range of genes and functions from many of the different eukaryotic taxa. In combination with novel emerging high-throughput technologies, we anticipate that this approach should contribute to exploring the functional diversity of the eukaryotic microbiota.

Isolation of multi-metal tolerant ubiquitin fusion protein from metal polluted soil by metatranscriptomic approach

Release of heavy metals into the soil pose a significant threat to the environment and public health because of their toxicity accumulation in the food chain and persistence in nature. The potential of soil microbial diversity of cadmium (Cd) contaminated site was exploited through functional metatranscriptomics by construction of cDNA libraries A (0.1-0.5u202fkb), B (0.5-1.0u202fkb), and C (1-4u202fkb) of variable size, from the eukaryotic mRNA. The cDNA library B was further screened for cadmium tolerant transcripts through yeast complementation system. We are reporting one of the transformants ycf1ΔPLBe1 capable of tolerating high concentrations of Cd (40u202fμM - 80u202fμM). Sequence analysis revealed that PLBe1 cDNA showed homology with ubiquitin domain containing protein fused with AN1 type zinc finger protein of Acanthameoba castellani. Further, this cDNA was tested for its tolerance towards other heavy metals such as copper (Cu), zinc (Zn) and cobalt (Co). Functional complementation assay of cDNA PLBe1 showed a range of tolerance towards copper (150u202fμM - 300u202fμM), zinc (10u202fmM - 12u202fmM) and cobalt (2u202fmM - 4u202fmM). This study promulgates PLBe1 as credible member of multi-metal tolerant gene in the eukaryotic soil microbial community and can be used as potential member to revitalise the heavy metal contaminated sites or can be used as a biomarker to detect heavy metal contamination in the soil environment.