Elena Martino

Solubilization of insoluble inorganic zinc compounds by ericoid mycorrhizal fungi derived from heavy metal polluted sites

Abstract Ericoid mycorrhizal fungi increase the ability of their host plants to colonize soils polluted with toxic metals, although the underlying mechanisms are not clearly understood. Two mycorrhizal strains of Oidiodendron maius isolated from contaminated soil were previously shown to tolerate high concentrations of toxic metals. We investigated further the biological mechanisms that may explain metal tolerance, focussing on the interactions between insoluble metal species and extracellular fungal metabolites. In particular, we demonstrate that fungal strains derived from polluted and unpolluted soils mobilize insoluble inorganic zinc compounds to different extents. Strains from polluted soils showed in fact little ability to solubilize Zn from both ZnO and Zn 3 (PO 4 ) 2 , whereas strains from unpolluted soils showed a higher solubilization potential. This different behaviour was confirmed when the solubilization abilities of a wider range of fungal strains (25 isolates) was examined. Induction of organic acids (malate and citrate) by the metal compounds was at least in part responsible for metal solubilization. Our results suggest that ericoid mycorrhizal strains from polluted and unpolluted soils may interact differently with metal compounds. We speculate that this may reflect specific strategies to maintain homeostasis of essential metals under different soil conditions.

Ericoid mycorrhizal fungi from heavy metal polluted soils: their identification and growth in the presence of zinc ions

Ericoid mycorrhizal fungi can alleviate heavy metal toxicity to their host plant, but the mechanisms that lie behind this increased tolerance are unknown. As a first step in the characterisation of two isolates of Oidiodendron maius from mycorrhizal roots of Vaccinium myrtillus growing in heavily contaminated soils, we investigated their taxonomic position, their mycorrhizal capabilities and their ability to grow in the presence of heavy metals. When growth was compared with isolates from non-polluted soils, a better performance was observed in the presence of increasing concentrations of zinc salts, especially at higher ion concentrations. The mechanisms of tolerance may include the production of mucilage and extracellular pigments.

Ericoid mycorrhizal fungi: some new perspectives on old acquaintances

Many ericaceous species colonize as pioneer plants substrates ranging from arid sandy soils to moist mor humus, in association with their mycorrhizal fungi. Thanks to the symbiosis with ericoid mycorrhizal fungi, ericaceous plants are also able to grow in highly polluted environments, where metal ions can reach toxic levels in the soil substrate. For a long time this mycorrhizal type has been regarded as an example of a highly specific interaction between plants and fungi. More recent studies have been challenging this view because some ericoid mycorrhizal endophytes seem also able to colonise plants from very distant taxa. A molecular approach has allowed the investigation of genetic diversity and molecular ecology of ericoid mycorrhizal fungi, and has revealed that ericaceous plants can be very promiscuous, with multiple occupancy of their thin roots. The molecular analysis of sterile morphotypes involved in this symbiosis has also led to deeper understanding of the species diversity of ericoid fungi. Genetic polymorphism of ericoid fungi is wider than previously thought, and often increased by the presence of Group I introns in the nuclear small subunit rDNA.

SOD1-Targeted Gene Disruption in the Ericoid Mycorrhizal Fungus Oidiodendron maius Reduces Conidiation and the Capacity for Mycorrhization

The genome sequences of mycorrhizal fungi will provide new opportunities for studying the biology and the evolution underlying this symbiotic lifestyle. The generation of null mutants at the wild-type loci is one of the best methods for gene-function assignment in the post-genomic era. To our knowledge, the generation of superoxide dismutase 1 (SOD1)-null mutants in the ericoid mycorrhizal fungus Oidiodendron maius is the first example of a gene-targeted disruption via homologous recombination in a mycorrhizal fungus. The disruption of OmSOD1 by Agrobacterium-mediated transformation resulted in the presence of oxidative stress markers, even in the absence of external superimposed stresses, and an increased sensitivity to reactive oxygen species (ROS)-generating substances, especially to menadione. A reduction in conidiation and in the percentage of mycorrhization of Vaccinium myrtillus roots was also observed. The latter findings establish the pivotal role of SOD1 as an important factor in the relationship between O. maius and its symbiotic partner. The lack of this ROS-scavenger may cause an imbalance in the redox homeostasis during host colonization and an alteration in the delicate dialogue between the fungus and its host plant.

Imaging mycorrhizal fungal transformants that express EGFP during ericoid endosymbiosis

Ericoid endomycorrhizal fungi form intracellular associations with the epidermal root cells of plants belonging to Ericales. In natural environments, these fungi increase the ability of their host plants to colonise soils polluted with toxic metals, although the underlying mechanisms are not clearly understood. Genetic transformation is a powerful tool to study the function of specific genes involved in the interaction of symbiotic fungi with the host plants and with the environment. Here, we investigated the possibility to genetically transform an ericoid endomycorrhizal strain. A metal tolerant mycorrhizal Oidiodendron maius strain isolated from a contaminated area was chosen to develop the transformation system. Two different protocols were used: protoplasts and Agrobacterium-mediated transformation. Stable transformants were obtained with both techniques. They remained competent for mycorrhizal formation and GFP-transformed fungi were visualised in planta. This is the first report of stable transformation of an ericoid endomycorrhizal fungus. The protocol set up could represent a good starting point for the identification of genes important in the ericoid mycorrhiza formation and in the understanding of how this symbiosis is established and functions. The success in the genetic transformation of this strain will allow us to better define its potential use in bioremediation strategies.

Cu,Zn superoxide dismutase and zinc stress in the metal-tolerant ericoid mycorrhizal fungus Oidiodendron maius Zn.

The sequence encoding a superoxide dismutase (SOD) was isolated from the cDNA library of a zinc-tolerant strain of the ericoid mycorrhizal fungus Oidiodendron maius, grown under zinc-stress conditions. Sequence homology to other SODs strongly suggests that it is a copper- and zinc-containing SOD. Functional complementation assays showed that the gene confers increased tolerance to zinc and copper stress to a Cu,ZnSOD-defective yeast mutant. Monitoring of transcript and protein levels following zinc stress suggests that OmSOD1 expression is controlled at the transcriptional level. The OmSod1 protein was found both in the cell extract and in the growth medium of viable fungal cultures. This is the first characterization of an extracellular Cu,ZnSOD in a mycorrhizal fungus. In nature, the presence of OmSod1 in the extracellular environment may also extend the protective role of this enzyme to the plant symbiont. This may be of particular interest from the perspective of using mycorrhizal fungi in bioremediation programmes.

Influence of zinc ions on protein secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungas Oidiodendron maius

A heavy metal tolerant strain of the ericoid mycorrhizal species Oidiodendron maius, isolated from soil heavily contaminated with zinc, was previously shown to tolerate high concentrations of zinc and cadmium ions in the growth medium. We have investigated some of the specific molecular responses of this fungal strain to the presence of increasing concentrations of zinc ions in the growth medium. In particular, we show that zinc ions induce a general change in the array of secreted proteins, with a shift towards the production of more basic, low molecular weight polypeptides. Some of these proteins were microsequenced and identified through homology search in databases. Among them are hydrolytic enzymes (nuclease, proteinase, lysozyme) and two superoxide dismutase isoforms. The latter are antioxidant enzymes known to play a role in heavy metal response in plants, animals and microorganisms.

Zinc ions alter morphology and chitin deposition in an ericoid fungus

A sterile mycelium PS IV, an ascomycete capable of establishing ericoid mycorrhizas, was used to investigate how zinc ions affect the cellular mechanisms of fungal growth. A significant reduction of the fungal biomass was observed in the presence of millimolar zinc concentrations; this mirrored conspicuous changes in hyphal morphology which led to apical swellings and increased branching in the subapical parts. Specific probes for fluorescence and electron microscopy localised chitin, the main cell wall polysaccharide, on the inner part of the fungal wall and on septa in control specimens. In Zn-treated mycelium, hyphal walls were thicker and a more intense chitin labelling was detected on the transverse walls. A quantitative assay showed a significant increase in the amount of chitin in metal-treated hyphae.

Morphological analysis of early contacts between pine roots and two ectomycorrhizal Suillus strains

Abstract Selection of ectomycorrhizal strains for application in forestry is mostly based on the evaluation of symbiotic performance in small-scale experiments. Two Suillus collinitus strains isolated from a Mediterranean and an alpine area were inoculated onto two pine tree species (Pinus pinea and P. nigra ssp. laricio var. corsicana) typical of these two environments. The early events during contact between the cell surfaces of plant and fungal partners were analysed morphologically using ultrastructural and immunocytochemical techniques. All four plant-fungus combinations led to a similar degree of mycorrhizal infection and to a similar colonization pattern. The first contact of fungal hyphae with root cap cells usually involved breakdown of the outermost electron-opaque layer of the plant cell walls. Hyphae further developed between this layer and the underlying wall strata. Ultrastructural observations revealed that S. collinitus strain J3-15-24, isolated from a Mediterranean area, induced a defence reaction in the roots of P. nigra, which grows typically in alpine areas. These observations suggest functional differences between the two fungal strains in their mycorrhizal capabilities.

Influence of heavy metals on production and activity of pectinolytic enzymes in ericoid mycorrhizal fungi

Ericoid mycorrhizal fungi increase the ability of their host plants to colonise soils polluted with heavy metal ions, although the mechanisms are not clearly understood. Two mycorrhizal mycelia of Oidiodendron maius isolated from contaminated soil were previously shown to tolerate high concentrations of heavy metal ions in the growth medium. The influence of zinc ions on the secretion and activity of polygalacturonase (PG), an extracellular enzyme that hydrolyses the pectin component of the plant cell walls, was investigated because of their significance during saprotrophic growth. Two major PG isoforms expressed both in the absence and in the presence of increasing concentrations of zinc ions were identified. The PG isoforms were purified from the tolerant Oidiodendron strains as well as from ericoid fungal isolates from non-polluted soils. Addition of increasing concentrations of Zn and Cd ions to the purified enzymes resulted in an increase of PG activity of both tolerant and non tolerant O. maius isolates at doses below 1 mM. By contrast, at the same metal ion concentrations PG activity of a sterile isolate from non-polluted soil was unaffected or slightly inhibited. We speculate that the response of PG to heavy metal ions may have been a pre-adaptive factor for the colonisation of polluted soils by O. maius.