Comparative genomics and expression levels of hydrophobins from eight mycorrhizal genomes.

Hydrophobins are small secreted proteins that are present as several gene copies in most fungal genomes. Their properties are now well understood: they are amphiphilic and assemble at hydrophilic/hydrophobic interfaces. However, their physiological functions remain largely unexplored, especially within mycorrhizal fungi. In this study, we identified hydrophobin genes and analysed their distribution in eight mycorrhizal genomes. We then measured their expression levels in three different biological conditions (mycorrhizal tissue vs. free-living mycelium, organic vs. mineral growth medium and aerial vs. submerged growth). Results confirmed that the size of the hydrophobin repertoire increased in the terminal orders of the fungal evolutionary tree. Reconciliation analysis predicted that in 41% of the cases, hydrophobins evolved from duplication events. Whatever the treatment and the fungal species, the pattern of expression of hydrophobins followed a reciprocal function, with one gene much more expressed than others from the same repertoire. These most-expressed hydrophobin genes were also among the most expressed of the whole genome, which suggests that they play a role as structural proteins. The fine-tuning of the expression of hydrophobin genes in each condition appeared complex because it differed considerably between species, in a way that could not be explained by simple ecological traits. Hydrophobin gene regulation in mycorrhizal tissue as compared with free-living mycelium, however, was significantly associated with a calculated high exposure of hydrophilic residues.

Bacterial communities associated with Brassica napus L. grown on trace element-contaminated and non-contaminated fields: a genotypic and phenotypic comparison.

Cultivable bacterial strains associated with field-grown Brassica napus L. (soil, rhizosphere and roots) from a trace elements (Cd, Zn and Pb) contaminated field and a non-contaminated control field were characterized genotypically and phenotypically. Correspondence analysis of the genotypic data revealed a correlation between soil and rhizosphere communities isolated from the same field, indicating that local conditions play a more important role in influencing the composition of (rhizosphere) soil bacterial communities than root exudates. In contrast, endophytic communities of roots showed a correlation between fields, suggesting that plants on the two fields contain similar obligate endophytes derived from a common seed endophytic community and/or can select bacteria from the rhizosphere. The latter seemed not very likely since, despite the presence of several potential endophytic taxa in the rhizosphere, no significant correlation was found between root and rhizosphere communities. The majority of Cd/Zn tolerant strains capable of phosphorus solubilization, nitrogen fixation, indole-3-acetic acid production and showing 1-aminocyclopropane-1-carboxylate deaminase capacity were found in the rhizosphere and roots of plants growing on the contaminated field.

Gene expression profiling of a Zn-tolerant and a Zn-sensitive Suillus luteus isolate exposed to increased external zinc concentrations.

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in different classes of expression pattern, were isolated and sequenced. Thirty-six of the represented genes showed homology to function-known genes, whereas 6 matched unknown protein coding sequences, and 102 were possibly novel. Although relatively few TDFs were found to be responsive to the different zinc treatments, their modulated expression levels may suggest a different transcriptional response to zinc treatments in both isolates. Among the identified genes that could be related to heavy-metal detoxification or the tolerance trait were genes encoding for homologues of a heat-shock protein, a putative metal transporter, a hydrophobin, and several proteins involved in ubiquitin-dependent proteolysis.

Phytostabilization of a metal contaminated sandy soil. I: Influence of compost and/or inorganic metal immobilizing soil amendments on phytotoxicity and plant availability of metals.

In a lysimeter set-up, compost addition to an industrial contaminated soil slightly reduced phytotoxicity to bean seedlings. The "Phytotoxicity Index" (on a scale from 1 to 4) decreased from 3.5 to 2.8. The same treatment also reduced metal accumulation in grasses: mean Zn, Cd and Pb concentrations decreased respectively from 623 to 135, from 6.2 to 1.3 and from 10.7 to <6 mg kg-1 dry weight. When combined with inorganic metal immobilizing amendments, compost had a beneficial effect on plant responses additional to the inorganic amendments alone. Best results were obtained when using compost (C)+cyclonic ashes (CA)+steel shots (SS). The "Phytotoxicity Index" decreased to 1.7, highest diversity of spontaneously colonizing plants occurred, and metal accumulation in grasses reduced to values for uncontaminated soils. Based on the first year evaluation, C+CA+SS showed to be an efficient treatment for amendment assisted phytostabilization of the contaminated Overpelt soil.

Phytostabilization of a metal contaminated sandy soil. II: Influence of compost and/or inorganic metal immobilizing soil amendments on metal leaching.

A lysimeter approach (under natural climatologic conditions) was used to evaluate the effect of four metal immobilizing soil treatments [compost (C), compost+cyclonic ashes (C+CA), compost+cyclonic ashes+steel shots (C+CA+SS)) and cyclonic ashes+steel shots (CA+SS)] on metal leaching through an industrially contaminated soil. All treatments decreased Zn and Cd leaching. Strongest reductions occurred after CA+SS and C+CA+SS treatments (Zn: -99.0% and -99.2% respectively; Cd: -97.2% and -98.3% respectively). Copper and Pb leaching increased after C (17 and >30 times for Cu and Pb respectively) and C+CA treatment (4.4 and >3.7 times for Cu and Pb respectively). C+CA+SS or CA+SS addition did not increase Cu leaching; the effect on Pb leaching was not completely clear. Our results demonstrate that attention should be paid to Cu and Pb leaching when organic matter additions are considered for phytostabilization of metal contaminated soils.

Copper-adapted Suillus luteus, a symbiotic solution for pines colonizing Cu mine spoils.

Natural populations thriving in heavy-metal-contaminated ecosystems are often subjected to selective pressures for increased resistance to toxic metals. In the present study we describe a population of the ectomycorrhizal fungus Suillus luteus that colonized a toxic Cu mine spoil in Norway. We hypothesized that this population had developed adaptive Cu tolerance and was able to protect pine trees against Cu toxicity. We also tested for the existence of cotolerance to Cu and Zn in S. luteus. Isolates from Cu-polluted, Zn-polluted, and nonpolluted sites were grown in vitro on Cu- or Zn-supplemented medium. The Cu mine isolates exhibited high Cu tolerance, whereas the Zn-tolerant isolates were shown to be Cu sensitive, and vice versa. This indicates the evolution of metal-specific tolerance mechanisms is strongly triggered by the pollution in the local environment. Cotolerance does not occur in the S. luteus isolates studied. In a dose-response experiment, the Cu sensitivity of nonmycorrhizal Pinus sylvestris seedlings was compared to the sensitivity of mycorrhizal seedlings colonized either by a Cu-sensitive or Cu-tolerant S. luteus isolate. In nonmycorrhizal plants and plants colonized by the Cu-sensitive isolate, root growth and nutrient uptake were strongly inhibited under Cu stress conditions. In contrast, plants colonized by the Cu-tolerant isolate were hardly affected. The Cu-adapted S. luteus isolate provided excellent insurance against Cu toxicity in pine seedlings exposed to elevated Cu levels. Such a metal-adapted Suillus-Pinus combination might be suitable for large-scale land reclamation at phytotoxic metalliferous and industrial sites.

AFLP-based assessment of the effects of environmental heavy metal pollution on the genetic structure of pioneer populations of Suillus luteus.

• The effects of environmental heavy metal pollution on the genetic structure of pioneer populations of the ectomycorrhizal basidiomycete Suillus luteus were assessed. • Sporocarps were collected from nine different locations and characterized by amplified fragment length polymorphism (AFLP) markers. Six of the sampling sites were contaminated with heavy metals and were dominated by tolerant individuals. Considerable genetic diversity was found within geographic subpopulations, but no reduction of the genetic diversity of populations inhabiting contaminated soils was observed. Neither did significant clustering of subpopulations inhabiting contaminated soils occur. Overall, the genetic differentiation between subpopulations was low, but Bayesian inference indicated the presence of two genetically differentiated clusters of individuals, which may correspond to different intercompatibility groups in S. luteus. • Heavy metal contamination seems to have a limited influence on the genetic structure of populations of S. luteus. Loss of diversity may have been prevented by sexual reproduction and rapid evolution of the tolerance trait or initial genetic bottlenecks may have been reduced by admixture and recurrent migration from surrounding populations colonizing noncontaminated soils.

Carbon and nitrogen allocation in ectomycorrhizal and non-mycorrhizal Pinus sylvestris L. seedlings.

We studied carbon and nitrogen allocation in mycorrhizal and non-mycorrhizal Scots pine (Pinus sylvestris L.) seedlings grown in a semi-hydroponic system with nitrogen as the growth limiting factor. Three ectomycorrhizal fungi were compared: one pioneer species (Thelephora terrestris Ehrh.: Fr.) and two late-stage fungi (Suillus bovinus (L.: Fr.) O. Kuntze, and Scleroderma citrinum Pers.). By giving all plants in each treatment the same amount of readily available nitrogen, we ensured that the external mycelium could not increase the total nitrogen content of the plants, thereby guaranteeing that any change in carbon or nitrogen partitioning was a direct effect of the mycorrhizal infection itself. Carbon and nitrogen partitioning were measured at an early and a late stage of mycorrhizal development, and at a low and a high N addition rate. Although mycorrhizal seedlings had a higher net assimilation rate and a higher shoot/root ratio than non-mycorrhizal seedlings, they had a lower rate of shoot growth. The high carbon demand of the mycobionts was consistent with the large biomass of external mycelia and the increased belowground respiration of the mycorrhizal plants. The carbon cost to the host was similar for pioneer and late-stage fungi. Above- and belowground partitioning of nitrogen was also affected by mycorrhizal infection. The external mycelia of Scleroderma citrinum retained 32% of the nitrogen supplied to the plants, thus significantly reducing nitrogen assimilation by the host plants and consequently reducing their growth rate. By contrast, the external mycelia of T. terrestris and Suillus bovinus retained less nitrogen than the mycelia of Scleroderma citrinum, hence we attributed the decreased growth rates of their host plants to a carbon drain rather than a nitrogen deficiency.

Reclamation of a bare industrial area contaminated by non-ferrous metals: physico-chemical and biological evaluation of the durability of soil treatment and revegetation.

In 1990, 3 ha of a highly metal polluted acid sandy soil at the site of a former pyrometallurgical zinc smelter was treated with a combination of beringite and compost; beringite is a substance that has a strong metal immobilization capacity. After soil treatment and sowing of a mixture of metal-tolerant Agrostis capillaris and Festuca rubra, a healthy vegetation cover developed. Five years later, an evaluation was made of soil physico-chemical parameters, potential phytotoxicity, floristic and fungal diversity and mycorrhizal infection of the plant community. Phytotoxicity was shown to be maintained at the low level observed immediately after soil treatment. The water-extractable metal fraction of the treated soil was up to 70 times lower compared to the non-treated soil. The vegetation was still healthy and regenerating by vegetative means and by seed. Diversity of higher plant species and saprophytic fungi was extremely low in the untreated area due to the high soil toxicity and the absence of metal tolerant ecotypes of plants and fungi. On the treated soil, in contrast, the species richness of higher plants was much higher; several perennial forbs which are not noted as metal tolerant had colonized the revegetated area. Most of these species belong to mycotrophic families so that the presence of a mycorrhizal network in the soil promotes their establishment. The ubiquity of the mycorrhizal fungi in the roots showed that a functioning ecosystem was establishing. In non-treated soil, the mycorrhizal infection rates of the roots were consistently lower during the whole growing season.