Elisa Zampieri

Is the Perigord black truffle threatened by an invasive species? We dreaded it and it has happened!

Invasive alien species are species introduced deliberately or unintentionally to areas outside their natural habitats. They can cause a significant irreversible environmental and socio-economic impact at genetic, species and ecosystem levels, and as claimed by Moore (2000), ‘throughout the world, exotic aliens are wreaking havoc’. The control of these invasive alien species has been discussed at international conventions, such as the Bern convention in 1979. Research on biological introductions has primarily focused on plant and animal invaders. Until now only a few studies have dealt with the spread of fungi through different continents and countries (Desprez-Loustau et al., 2007), largely because of a lack of scientific knowledge of fungal biodiversity and ecology. Reports have mostly focused on the introduction of agronomically important plant pathogenic fungi (above all, rusts and Ascomycetes) and pseudofungi (Stramenopila and Peronosporomycetes), and their invasion routes (Desprez-Loustau et al., 2007). The most common way of introducing fungal pathogens is through the movement of infected planting stock or infested wood (Coetzee et al., 2001). The success of invasive fungal pathogens in these new environments might be explained by an increased aggressiveness towards new host species that have not had an opportunity to evolve resistance. The ecological impacts of the intentional/unintentional introduction of fungal species, apart from plant pathogens, have been largely ignored (Schwartz et al., 2006). Regarding saprotrophic taxa, in-depth studies have only been carried out with a few species, for example Coprinopsis stangliana (Bougher, 2006), and some woodchip fungi (Shaw et al., 2004). As far as ectomycorrhizal fungi are concerned, the accidental introduction and spread of Amanita phalloides (Pringle & Vellinga, 2006), Boletus edulis s.l. (Hall et al., 1998) and Eucalyptus-associated species, such as Pisolithus spp. (Martin et al., 2002), have been carefully studied and monitored. These symbiotic fungi were accidentally introduced when allochthonous trees were planted for agriculture and reforestation (Hall et al., 1998; Martin et al., 2002). The deliberate movement of nonindigenous mycorrhizal fungal species and strains is a phenomenon occurring with increasing frequency as a consequence of the use of beneficial soil organisms to improve horticulture (Azcon-Aguilar & Barea, 1997), bioremediation (Leyval et al., 2002), reforestation (Duponnois et al., 2005) and edible fruit body production (Hall et al., 1998; Hall et al., 2003). The impact of all these introductions on the overall native resident fungal communities is not well understood, and scant attention has been paid to the ecological impact of transporting nonpathogenic fungi across continents (with a few exceptions, e.g. Selosse et al., 1998). Monitoring efforts are therefore urgently needed to track the spreading pattern of introduced fungi.

Soil analysis reveals the presence of an extended mycelial network in a Tuber magnatum truffle-ground

Truffles are hypogeous ectomycorrhizal fungi. They belong to the genus Tuber and are currently considered a hot spot in fungal biology due to their ecological and economic relevance. Among all the species, Tuber magnatum is the most appreciated because of its special taste and aroma. The aim of this work was to set up a protocol to detect T. magnatum in soil and to assess its distribution in a natural truffle-ground. We used the beta-tubulin gene as a marker to identify T. magnatum in the soil. This gene allowed us to trace the distribution of the fungus over the entire truffle-ground. Tuber magnatum was found, in one case, 100 m from the productive host plant. This study highlights that T. magnatum mycelium is more widespread than can be inferred from the distribution of truffles and ectomycorrhizas. Interestingly, a new haplotype - never described from fruiting body material - was identified. The specific detection of T. magnatum in the soil will allow to unravel the ecology of this fungus, following its mycelial network. Moreover, this new tool may have practical importance in projects aimed to increase large-scale truffle production, checking for T. magnatum persistence in plantations.

The Perigord black truffle responds to cold temperature with an extensive reprogramming of its transcriptional activity.

The Tuber melanosporum genome has been analysed with the aim of identifying and characterizing the genes involved in the environmental stress response. A whole genome array (7496 genes/probe) was used to verify the fungal transcriptional profiling upon a cold temperature period (7 days at 4 °C). A total of 423 genes resulted to be differentially expressed in a significant manner (>2.5-fold; p-value<0.05) in the mycelia exposed to cold, compared to the control ones: 187 of these genes were up-regulated, while 236 were down-regulated. Sixty-six and fifty-one percent, respectively, of the up- or down-regulated transcripts had no KOG classification and were clustered as unclassified proteins, which was the most abundant category in the both up- and down-regulated genes. A gene subset, containing a range of biological functions, was chosen to validate the microarray experiment through quantitative real time PCR (qRT-PCR). The analysis confirmed the array data for 16 out of 22 of the considered genes, confirming that a cold temperature period influences the truffle global gene expression. The expressed genes, which mostly resulted to be genes for heat shock proteins (HSPs) and genes involved in cell wall and lipid metabolism, could be involved in mechanisms, which are responsible for fungal adaptation. Since truffle ascomata develop during the winter period, we hypothesize that these differentially expressed genes may help the truffle to adapt to low temperatures and/or perceive environmental signals that regulate the fructification.

Identification of internal transcribed spacer sequence motifs in truffles: a first step toward their DNA bar coding.

ABSTRACT This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (≤50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.

Specific regions in the Sod1 locus of the ericoid mycorrhizal fungus Oidiodendron maius from metal-enriched soils show a different sequence polymorphism

The huge diversity of fungi may reflect both the heterogeneity of the niches they occupy and the diverse stresses they must cope with. In order to investigate the genetic and functional diversity in the ericoid mycorrhizal fungus Oidiodendron maius subjected to heavy metal stress, we isolated O. maius strains from a serpentine site naturally enriched by heavy metals. Despite the high Cr and Ni soil concentrations, a high level of diversity was found in the serpentine fungal community. The growth of these isolates in the presence of different metal contaminants identified some tolerant strains, suggesting a site-specific adaptation. To investigate within-species gene divergence in stressful environments, we then compared the sequence polymorphism of a neutral (internal transcribed spacer) and a functional (Cu,ZnSOD) gene in O. maius isolates derived from the serpentine site, from a site heavily polluted with industrial wastes and from unpolluted sites. For all isolates tested, the polymorphism was higher in the nucleotide sequence of the functional gene. However, when compared with isolates from the serpentine area, isolates from industrially polluted sites showed a significantly higher polymorphism in the Cu,ZnSOD promoter region, suggesting that environmental stress may influence the rate of mutations in specific regions of the Sod1 locus.

Soil metaproteomics reveals an inter-kingdom stress response to the presence of black truffles

For some truffle species of the Tuber genus, the symbiotic phase is often associated with the presence of an area of scant vegetation, commonly known as the brûlé, around the host tree. Previous metagenomics studies have identified the microorganisms present inside and outside the brûlé of a Tuber melanosporum truffle-ground, but the molecular mechanisms that operate in this ecological niche remain to be clarified. To elucidate the metabolic pathways present in the brûlé, we conducted a metaproteomics analysis on the soil of a characterized truffle-ground and cross-referenced the resulting proteins with a database we constructed, incorporating the metagenomics data for the organisms previously identified in this soil. The soil inside the brûlé contained a larger number of proteins and, surprisingly, more proteins from plants, compared with the soil outside the brûlé. In addition, Fisher’s Exact Tests detected more biological processes inside the brûlé; these processes were related to responses to multiple types of stress. Thus, although the brûlé has a reduced diversity of plant and microbial species, the organisms in the brûlé show strong metabolic activity. Also, the combination of metagenomics and metaproteomics provides a powerful tool to reveal soil functioning.

PCR primers specific for the genus Tuber reveal the presence of several truffle species in a truffle-ground

Truffles are hypogeous Ascomycete fungi belonging to the genus Tuber and forming fruiting bodies highly prized for their taste and aroma. The identification of the genus Tuber and its species is important to investigate their ecology and avoid fraud in the food market. As genus-specific primers are not available, the aims of this work were (1) to assess the usefulness of the beta-tubulin gene as a DNA barcoding region for designing Tuber genus-specific primers, (2) to test the primers on a range of fruiting bodies, representing a large part of truffle biodiversity and (3) to check their ecological usefulness, applying them to truffle-ground soil. The new primers designed on the beta-tubulin gene were specific to the Tuber genus in nested PCR. When applied to DNA from soils, they gave a positive signal for 23 of 32 soils. Phylogenetic analysis confirmed that the bands corresponded to Tuber and that at least five Tuber species were present in the truffle-ground. beta-tubulin was found to be a good barcoding region for designing Tuber genus-specific primers, detecting a high Tuber diversity in a natural environment. These primers will be useful for understanding truffle ecology and for practical needs in plantation management.

Identification of genes differentially expressed during the interaction between the plant symbiont Suillus luteus and two plant pathogenic allopatric Heterobasidion species

The effects of biological invasions by non-native species have been widely studied in terms of environmental, economic, and human health impacts. However, little is known about the consequences that non-native plant pathogens may determine on host plant symbionts, such as ectomycorrhizal (ECM) fungi. In this study, interactions between Suillus luteus, an ECM fungus of pine trees, and the allopatrically differentiated fungal pathogens of pines Heterobasidion irregulare and H. annosum were investigated in dual culture by morphological and gene expression analyses. Growth of S. luteus was inhibited by both Heterobasidion species, but based on statistical analysis, growth inhibition was due to the isolate rather than to the species. The expression analysis on genes related to cell wall hydrolytic enzymes and hydrophobins, putatively involved in the fungus–fungus interaction, allowed to identify significantly up- and down-regulated genes both in the symbiont and in the pathogens. Based on the transcript analysis, it was not possible to distinguish the impact of the two pathogenic species on the ECM fungus. The only exception was a S. luteus gene coding for a putative chitinase (SlGH18_8356) that was found to be differentially regulated during interaction with H. irregulare compared to H. annosum.

Genomic suppression subtractive hybridization as a tool to identify differences in mycorrhizal fungal genomes.

Characterization of genomic variation among different microbial species, or different strains of the same species, is a field of significant interest with a wide range of potential applications. We have investigated the genomic variation in mycorrhizal fungal genomes through genomic suppressive subtractive hybridization. The comparison was between phylogenetically distant and close truffle species (Tuber spp.), and between isolates of the ericoid mycorrhizal fungus Oidiodendron maius featuring different degrees of metal tolerance. In the interspecies experiment, almost all the sequences that were identified in the Tuber melanosporum genome and absent in Tuber borchii and Tuber indicum corresponded to transposable elements. In the intraspecies comparison, some specific sequences corresponded to regions coding for enzymes, among them a glutathione synthetase known to be involved in metal tolerance. This approach is a quick and rather inexpensive tool to develop molecular markers for mycorrhizal fungi tracking and barcoding, to identify functional genes and to investigate the genome plasticity, adaptation and evolution.

Ectomycorrhizal Fungi and Their Applications

Ectomycorrhizal (ECM) fungi form association with relatively small number of plants that dominate boreal, temperate, Mediterranean, and some subtropical forest ecosystems. These plant species have been able to acquire metabolic capabilities through symbioses with ECM fungi, thus improving their mineral nutrition and growth in several ecological niches. Mycorrhizal fungi can also play several other important ecological roles, including the protection of plants from abiotic and biotic stresses. Several “targeted” metagenomic projects have been carried out, or are now in progress, in order to identify the fungal communities in soil, including ECM fungi, which are present in various habitats (e.g., forest and truffle-ground soils, etc.). ECM fungi, which are important both because of their economic value as edible fungi (i.e., truffles, boletes) and because of their application in reforestation projects, are the subject of this chapter, in which the recent advances in ECM fungal communities are reviewed, focusing mainly on the applicative aspects related to the use of these fungi.